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Physical Insights into Chemistry, Catalysis, and Interfaces

Interplay of Affinity and Surface Tethering in Protein Recognition
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  • Ali Imran
    Ali Imran
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
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  • Brandon S. Moyer
    Brandon S. Moyer
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
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  • Aaron J. Wolfe
    Aaron J. Wolfe
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
    Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
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  • Michael S. Cosgrove
    Michael S. Cosgrove
    Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
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  • Dmitrii E. Makarov
    Dmitrii E. Makarov
    Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States
    Oden Institute for Computational Engineering and Sciences, University of Texas at Austin, Austin, Texas 78712, United States
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    Orcidhttps://orcid.org/0000-0002-8421-1846
  • Liviu Movileanu*
    Liviu Movileanu
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, New York 13244, United States
    The BioInspired Institute, Syracuse University, Syracuse, New York 13244, United States
    *Phone: 315-443-8078. Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-2525-3341
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The Journal of Physical Chemistry Letters

Cite this: J. Phys. Chem. Lett. 2022, 13, 18, 4021–4028
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https://doi.org/10.1021/acs.jpclett.2c00621
Published April 29, 2022

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Abstract

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Surface-tethered ligand–receptor complexes are key components in biological signaling and adhesion. They also find increasing utility in single-molecule assays and biotechnological applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the association of ligand–receptor complexes, experimentally proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissociation. Notably, the rate constants measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand–receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomolecular recognition, intrinsically disordered proteins, and biosensor technology.

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Tethered ligand–receptor complexes are common in protein recognition (1,2) and cellular adhesion. (3) Surface-bound ligand–protein complexes are also the basis for biotechnological applications, such as biosensors (4−9) and cell-targeted therapeutic proteins, (10,11) as well as for single-molecule techniques that probe the dynamics and thermodynamics of protein binding. (12−16) Yet, how the presence of spatial constraints imposed by the surface and/or the tether affects the thermodynamics and, especially, kinetics of binding is largely an open experimental question. Most of the current insight into this topic comes from theoretical (17−21) and computational (10,22−24) studies. However, experimental examinations of tethered ligand–protein interactions are mostly limited to measuring macroscopic intermolecular forces, (25−28) equilibrium dissociation constants, (29) and effective protein concentrations. (29,30)

In contrast to the earlier experimental work, this study focuses on the question of how the kinetics of binding and unbinding is altered by the tethering of one of the binding partners to a surface. To this end, we measure the real-time kinetics of tethered ligand–receptor complexes using surface immobilization-based sensing approaches. In our case, the receptor is WD40 repeat protein 5 (WDR5), (31,32) a chromatin-associated hub that is primarily known for its regulatory role in histone methylation. (33,34) The 334-residue WDR5 features a seven-bladed β propeller circular structure and a central cavity. The WDR5 cavity hosts the binding site for the WDR5-interaction (Win) motif of human mixed lineage leukemia (MLL/SET1) methyltransferases, also named the Win binding site. We examined details of the interactions of five 14-residue Win-motif peptide ligands of SET1 proteins (SET1Win ligands; Table S1 and Supplemental Methods) (35,36) with WDR5 via its Win binding site. SET1Win ligands were chemically attached to a streptavidin-coated surface. Either a three-residue short peptide tether (ST-SET1Win ligands; Figure 1a) or a nine-residue long peptide tether (LT-SET1Win ligands; Figure 1b) was inserted between the biotinylated attachment site of the SET1Win ligand to the surface and the SET1Win sequence. In this way, the binding kinetics of the WDR5–SET1Win complex was probed by using biolayer interferometry (BLI). (37) The association and dissociation phases of the tethered ligand–receptor complex were discriminated optically by using changes in the interference pattern of reflected light waves at the sensor surface. Hence, these interactions were monitored by using WDR5-containing and WDR5-free assay buffers, respectively. Tethered ligand–receptor interactions were also evaluated by using Win binding site-directed WDR5 mutants (Table S2 and Supplemental Methods). To further examine the binding kinetics in the absence of restraining tethers, WDR5 proteins were immobilized on the surface plasmon resonance (SPR) sensors (38) (no tether, NT-SET1Win ligands; Figure 1c).

Figure 1

Figure 1. WDR5 protein interacting with the SET1Win peptide ligands under different conditions. WDR5 is shown in orange, while SET1Win ligands are shown in magenta. Bound interacting partners are shown in blue. Lightly colored receptors and ligands indicate interacting partners in the background. (a) Biotinylated ST-SET1Win ligands were chemically attached onto a streptavidin-coated biolayer interferometry (BLI) sensor surface. Either WDR5 proteins or one of its mutants was freely movable in solution. (b) The same system as in (a), but with LT-SET1Win ligands. (c) Either WDR5 proteins or one of its mutants was immobilized onto a surface plasmon resonance (SPR) chip surface, whereas the NT-SET1Win ligands were freely movable in solution.

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We obtained the real-time kinetics of five SET1Win peptide ligands (MLL2Win, MLL3Win, MLL4Win, SETd1AWin, and SETd1BWin) with four WDR5 proteins (wild-type and 3 mutants of the Win binding site, P216L, F133L, and S218F) using ST and LT constraints (Figures S1 and S2,Tables S3–S5). Later, we validated the outcomes of this study using S175L, a fourth WDR5 mutant of unknown affinity. Interestingly, the association rate constants, ka, acquired with LT-SET1Win ligands (ka-LT) were on average higher than those corresponding values recorded with ST-SET1Win ligands (ka-ST) (Figure 2a and Table S6). To explain this observation, we considered the general framework of diffusion-controlled reactions, (39,40) which gives the following association rate constant:

ka=(kD−1+kR−1)−1
(1)
where kR is the reaction-controlled rate constant and
kD=4πDrela
(2)
is the diffusion-controlled rate constant that depends on the relative diffusion coefficient of the two reacting species, Drel, and on a “geometric” parameter, a. Here, a is the contact distance or capture radius between the centers of the two interacting partners considered as spheres. In the limit kR ≫ kD, the association is purely diffusion controlled and ka ≈ kD. (41)Equation 2 may be loosely interpreted as the rate constant of the association process happening instantaneously upon the reactants diffusing into a favorable relative configuration. This configuration is characterized by a linear length scale, a. Notably, simple dimensionality arguments require that the diffusion-controlled rate constant, kD, must be of the form of eq 2. Hence, eq 2 can be viewed as the definition of the effective “target” size of the diffusion-controlled reaction.

Figure 2

Figure 2. Scatter plots of kinetic and equilibrium constants for ST-SET1Win and LT-SET1Win ligands. (a) Association rate constants ka-ST of ST-SET1Win-WDR5 complexes plotted against association rate constants ka-LT of LT-SET1Win-WDR5 complexes. Points above the blue line correspond to complexes with faster association rate constants for ST-SET1Win ligands, while points below correspond to interactions with slower association rate constants for ST-SET1Win ligands. (b) Dissociation rate constants kd-ST of ST-SET1Win–WDR5 complexes plotted against dissociation rate constants kd-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to complexes with faster dissociation rate constants for ST-SET1Win ligands. (c) Equilibrium dissociation constants KD-ST of ST-SET1Win–WDR5 complexes plotted against equilibrium dissociation constants KD-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to less stable complexes with ST-SET1Win ligands. m indicates the slopes of linear fits in (b) and (c). Data represent mean ± s.d. which resulted from three independent BLI sensorgrams.

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There are two notable examples of eq 2. First, Smoluchowski has obtained a formula for the diffusion-controlled rate constant, where the association process between two spherically symmetrical reactants takes place whenever their distance reaches the “capture radius” value a. (42,43) Second, Berg and Purcell derived a formula for the rate constant of the process where a freely diffusing particle hits a patch on a planar wall, with a being the linear size of the patch. (44) The Berg and Purcell scenario can be viewed as a prototype for the system studied here, as one of the reactants is surface immobilized.

It should be noted that the length parameter, a, generally depends on the interaction between the reactants. (39,40) Therefore, the parameter a is not purely geometric. (45,46) For example, for the model where the ligand and receptor are approximated as spheres interacting via a centrosymmetric potential, U(r), the diffusion-controlled rate constant to reach a geometric contact distance R is given by eq 2, with a defined as (39)

a=[∫R∞eU(r)/kBTr−2dr]−1
(3)
where kBT is the thermal energy and r is the intersphere distance. Unless U(r) = 0, a is different from the pure geometric capture radius R. Consistent with intuition, for example, attractive electrostatic interaction increases the apparent value of a. Rotational diffusion and site-specific physical restrains of interacting molecules may further affect the apparent value of a. The sensitivity of the effective capture radius, a, to the interaction energy explains, at least in part, the small changes in the ka between different SET1Win peptide ligands (Figure 2a and Table S3).

Equipped with these ideas, we consider the difference between the cases of ST- and LT-SET1Win ligands. The much smaller, surface-attached SET1Win ligand diffuses rapidly, with a diffusion coefficient DSET1Win ≫ DWDR5. Diffusion of the SET1Win ligand occurs around its attachment point within a certain volume, which depends on the tether length. This suggests a simple model of association, as follows. Like in Berg and Purcell’s model, (44) the surface-attached SET1Win ligand appears as target with a characteristic size, a, to a freely diffusing WDR5. Because of the complicated geometry of the system, it is challenging to derive a simple expression for a. LT-SET1Win can deviate further from the attachment point than ST-SET1Win. Therefore, LT-SET1Win is a larger “target” for the WDR5 than ST-SET1Win (i.e., aLT > aST), so the association rate constant for LT-SET1Win, ka–LT, is higher than that for ST-SET1Win, ka–ST, as observed in Figure 2a. Note, however, that this picture is expected to break down in the limit of long tethers where further increase of the tether length results in a larger search volume that has to be explored by the binding partners, reducing the overall association rate constant. Indeed, as recently discussed by Misiura and Kolomeisky, (45) the dependence of the association rate constant on the tether length is nonmonotonic, with the maximum association speedup occurring at an intermediate tether length.

The association speedup induced by a longer tether found here is an experimental validation of the “fly-casting association mechanism”, which was proposed earlier by Wolynes and co-workers on theoretical grounds and computational analysis (47−49) and discussed later by others. (13,45,50−54) This mechanism explains how intrinsically disordered proteins with random-coil conformations can bind faster to their targets. (12,55) Because of the geometric nature of the parameter a, it is expectable that the ratio of a values for LT-SET1Win and ST-SET1Win, aLT/aST, is nearly the same for all SET1Win ligands. Indeed, we observe a linear correlation between the association rate constants for LT-SET1Win and ST-SET1Win, ka–LT and ka–ST, respectively (Figure 2a). But recalling that the parameter a also depends on the energetics of the interactions, deviations from a perfectly linear correlation are not surprising.

In contrast to the association rate constants, the dissociation rate constants for ST-SET1Win ligands, kd-ST, were consistently higher than those for LT-SET1Win ligands, kd-LT (Figure 2b; Tables S7 and S8). Furthermore, kd-ST and kd-LT values closely followed a proportionality relationship. To explain these observations, we start with the Arrhenius law for the unimolecular dissociation process: (41)

kd=νexp(−ΔGakBT)
(4)
where ν is a prefactor and ΔGa is the activation free energy, which is determined by the strength of cohesive interactions between SET1Win and WDR5. It is known that a microscopic object (e.g., a Brownian particle) tethered to a surface via a flexible polymer tether experiences a repulsive net force that pushes it away from the surface even when the surface is perfectly neutral. This is a typical situation in a single-molecule experiment, where a microscopic bead is anchored to a surface. This force is “entropic” in its nature, originating from the fact that the bead has more space available when it is further away from the surface. For example, if the tether length becomes very small, then this exclusion-volume effect is significant, resulting in a steric wall repulsion of the bead from the surface. (28) The properties of this force have been theoretically studied by Segall and co-workers, (56) who showed that it is roughly inversely proportional to the distance from the surface. Our real-time binding kinetics experiments with ST-SET1Win involve a three-residue tether. This means that our ST-SET1Win ligand–WDR5 receptor complex, whose size is ∼4.5 nm, is constrained to statistically fluctuate at a distance shorter than ∼1 nm from the surface. Here, we speculate that under these conditions the exclusion-volume effect of the tethered complex pushes WDR5 away from the surface and thus from SET1Win as well. In this way, the steric wall repulsion enhances the dissociation of WDR5 receptors from SET1Win ligands by lowering their dissociation barrier.

The simplest approximate description of this mechanochemical effect for the dissociation rate constant, kd, is the Eyring–Zhurkov–Bell formula: (57)

kd=νexp(−ΔGa−fΔxkBT)=kd0exp(fΔxkBT)
(5)
where kd0 is kd at f = 0. Here, f is the magnitude of the force, and Δx is an activation length. Hence, kd0 is the dissociation rate constant in the absence of the surface. Clearly, the force f for ST-SET1Win, fST, is higher than that for LT-SET1Win, fLT. Therefore, the dissociation rate constant for ST-SET1Win, kd–ST, is greater than that for LT-SET1Win, kd–LT, as observed in Figure 2b. Assuming that the activation length Δx, being again a geometric parameter, is approximately the same for different constructs, the ratio of the two dissociation rate constants should be close to a constant. This should happen even though the rate constants themselves may vary considerably because of the variation of the activation free energy, ΔGa, and to exponential sensitivity of the dissociation rate constant to the energetics of interaction. Indeed, this is what we observe in Figure 2b. Despite almost 2 orders of magnitude variation between the individual kd constants for each construct, kd–ST and kd–LT remain proportional to each other. Note that the ka constants for the same constructs vary within a much narrower range, within a maximum factor of ∼4, supporting the above proposal that the association process is near the diffusion-controlled limit and thus less sensitive to energetics.

These results suggest that the length of the tether plays a significant role in modulating the interactions of the SET1Win–WDR5 complex. An increased physical constraint as a result of a decreased tether length not only reduces the rate constant of complex formation, as established earlier, but also substantially decreases the stability of the complex. Consequently, the overall impact of reducing the tether length is an increase in KD (Figure 2c; Tables S9 and S10). Changes observed for ka should normally be independent from those noted for kd because the mechanisms of changing the corresponding activation free energies are different. Indeed, we observed no correlation between the ka and kd values (Figures S3 and S4).

We then measured the kinetic rate constants for 20 ligand–receptor complexes using unrestricted conditions (no tether, NT-SET1Win ligands) (Figure S5, Tables S11–S13). In this case, BLI was not used because it does not have a satisfactory sensitivity to reliably detect a short-peptide binding to the surface. The SPR, (38) with its greater sensitivity, was a more effective choice for this case. Accumulation of ligand–receptor complexes onto the surface of the SPR sensor was monitored by changes in the refractive index. Therefore, WDR5 was immobilized onto the surface of the SPR chips (Figure 1c), and the association and dissociation phases were probed in real time. As established by our previous work, (58) the ka values for NT-SET1Win ligands were substantially greater than those for LT-SET1Win ligands (Figure 3a and Table S14). This significant difference is due to the increased translational and rotational diffusion coefficients of NT-SET1Win ligands relative to WDR5 and its derivatives. Moreover, our previous work (58) also showed, by comparison with values obtained from fluorescence polarization (FP) spectroscopy, that immobilizing WDR5 onto the SPR sensor surface does not impact its functional integrity. Let us assume that DNT–SET1Win and DWDR5 are the translational diffusion coefficients of NT-SET1Win and WDR5, respectively. For applying eq 2 to this problem, one now has to consider that DNT–SET1Win ≫ DWDR5 because either WDR5 or one of its derivatives was immobilized on the sensor surface. Therefore, the unrestrained NT-SET1Win was responsible for the diffusion-mediated mutual approach of the reacting species, so Drel ≈ DNT–SET1Win. Again, eq 2 predicts proportionality between ka–NT and ka–LT, as noted in Figure 3a, with the ratio of the two roughly equal to the ratio of SET1Win’s and WDR5’s diffusion coefficients.

Figure 3

Figure 3. Scatter plots of kinetic and equilibrium constants for NT-SET1Win and LT-SET1Win ligands. (a) Association rate constants ka-NT of NT-SET1Win–WDR5 complexes plotted against association rate constants ka-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to interactions with faster association rate constants for NT-SET1Win ligands. (b) Dissociation rate constants kd-NT of NT-SET1Win–WDR5 complexes plotted against dissociation rate constants kd-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to interactions with faster dissociation rate constants for NT-SET1Win ligands. (c) Equilibrium dissociation constants KD-NT values of NT-SET1Win–WDR5 complexes plotted against equilibrium dissociation constants KD-LT of LT-SET1Win–WDR5 complexes. Points below the blue line correspond to more stable complexes with LT-SET1Win ligands. m indicates the slopes of linear fits in all panels. Data represent mean ± s.d. which resulted from three independent BLI sensorgrams.

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Remarkably, the kd values using NT-SET1Win and LT-SET1Win ligands were closely similar (Figure 3b and Table S15). Our interpretation of this finding is in terms of eq 5. In the case of LT-SET1Win ligands, but not for ST-SET1Win ligands, the repulsive force f is negligible as the complex is far enough from the surface. Hence, the dissociation rate constant is near that value corresponding to the zero-force limit, kd0, which is the dissociation rate constant for NT-SET1Win ligands, kd–NT. In other words, at long enough tether lengths, the experimental system approaches that of NT-SET1Win ligands in terms of the dissociation rate constant, kd. Therefore, the equilibrium dissociation constant, KD, of the ligand–receptor complex becomes larger as we go from NT-SET1Win ligands to LT-SET1Win ligands (Figure 3c; Tables S16 and S17). Moreover, the differential free energy of the ligand–receptor complex formation, ΔΔG, for NT-SET1Win ligands with respect to LT-SET1Win ligands is in the range −0.3 through −1.5 kcal/mol. The primary contribution to this change results from the considerable increase in the ka in the absence of the tether. This shows how the attachment of a binding partner to a surface influences the overall dynamic equilibrium of the interaction. In our case, the effect is substantial given the large difference in size between the two binding partners. Even though for NT-SET1Win ligands the WDR5 is restricted to the surface, the comparison between similar restriction and steady-state fluorescence polarization (FP) data of freely interacting SET1Win and WDR5 in solution shows that this condition can be thought as that of an unrestricted interaction. (58)

In Figure 4a, we illustrate a qualitative comparison of the free energy landscapes that correspond to NT-SET1Win, ST-SET1Win, and LT-SET1Win ligands. For short and long tethers, the presence of the flexible tether reduces the association rate constant of the SET1Win–WDR5 complex with respect to that in the absence of the tether (Figure S6). Further increase in the kd-ST with respect to kd-LT (Figure S7) due to repulsion forces of WDR5 proteins from the sensor surface explains the relative increase in the normalized values (KD-ST/KD-NT) > (KD-LT/KD-NT) (Figure 4b–e). Because there are linear correlations between measured affinities of various SET1Win–WDR5 pairs with specified constraints, we can advantageously utilize these findings to predict the kd and KD for a given tethered ligand–receptor complex. To demonstrate this, we examined the interactions of SET1Win ligands with S175L, a WDR5 derivative, whose single-site mutation is located within the Win binding site. Using the kinetic and equilibrium parameters measured for NT-SET1Win–S175L interactions via SPR (Tables S12 and S13), we established the proportionality relationships with their corresponding parameters for ST-SET1Win ligands (Figure S8). Remarkably, our experimental determinations of kd-ST for S175L against five ST-SET1Win ligands are closely similar to corresponding anticipated values (Table 1). Furthermore, using the same method, we demonstrate the predictive power of this approach for the KD-ST values (Table 2). Therefore, the binding affinity of tethered ligand–receptor interactions can be precisely modulated by changing the tether length (Figure S9).

Table 1. Predicted and Experimental Values of the kd-ST for S175L Interacting with ST-SET1Wina
parameterSET1Winpredicted values ×103 (s–1)experimental values ×103 (s–1)
kd-STMLL2Win14 ± 112 ± 1
 MLL3Win36 ± 128 ± 1
 MLL4Win190 ± 10180 ± 10
 SETd1AWin300 ± 10160 ± 10
 SETd1BWin13 ± 15.4 ± 0.2
a

kd-ST are the dissociation rate constants corresponding to ST-SET1Win ligands. Predicted values of kd-ST were obtained using the proportionality relationship between kd-ST and kd-NT (Figure S8) and the experimentally determined values of kd-NT (Table S12). Triplicate kd-NT values were used to calculate corresponding kd-ST values by linear interpolation. Values indicate mean ± s.d., which were calculated by using these triplicates.

Table 2. Predicted and Experimental Values of the KD-ST for S175L Interacting with ST-SET1Wina
parameterSET1Winpredicted values ×109 (M)experimental values ×109 (M)
KD-STMLL2Win150 ± 10360 ± 30
 MLL3Win270 ± 10810 ± 90
 MLL4Win2800 ± 1008500 ± 300
 SETd1AWin5500 ± 2002900 ± 100
 SETd1BWin110 ± 10110 ± 6
a

KD-ST are the equilibrium dissociation constants corresponding to ST-SET1Win ligands. Predicted values of KD-ST were obtained using the proportionality relationship between KD-ST and KD-NT (Figure S8) and the experimentally determined values of KD-NT (Table S13). Triplicate KD-NT values were used to calculate corresponding KD-ST values by linear interpolation. Values indicate mean ± s.d., which were calculated by using these triplicates.

Figure 4

Figure 4. 3D plots and contour maps of normalized KD constants. (a) Qualitative free energy landscapes of SET1Win–WDR5 interactions when NT-SET1Win (NT), ST-SET1Win (ST), and LT-SET1Win (LT) peptide ligands were used. Vertical lines 1, 2, and 3, which are marked in cyan, indicate the differential free energy barriers due to unrestrained diffusion of the ligand, fly casting mechanism, and repulsion entropic forces of the receptor from the sensor surface, respectively. (b) Bar graph and (c) contour map of KD-ST values for the interaction of ST-SET1Win ligands, with WDR5 and its mutants, divided by their corresponding KD-NT values measured with the corresponding NT-SET1Win ligands. (d) Bar graph and (e) contour map of KD-LT values for the interaction of LT-SET1Win ligands, with WDR5 and its mutants, divided by their corresponding KD-NT values measured with the corresponding NT-SET1Win ligands. KD-ST and KD-LT for MLL4Win–F133L interactions could not be quantitatively determined by using BLI measurements. These data points are colored in black.

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In summary, we provide compelling experimental evidence for the fly casting mechanism of association between surface-attached peptide ligands and their receptors. The observed speedup in the association rate, ka, when using a longer tether is rather modest for the tether lengths employed here, which agrees with previous computational work. (47) We also found that the dissociation rate constant, kd, was greater in the case of a short tether length as a result of steric wall repulsion forces acting on the receptor pulling it away from the surface. Accordingly, this resulted in a weakened interaction of the tethered ligand–protein complex. As a longer tether accelerates the association but decelerates the dissociation, the binding affinity of the ligand–receptor complex is greater at increased tether lengths. From a practical point of view, our experimental approach can be used to predict dissociation rate constants and binding affinities of ligand–protein interactions for specified physicochemical properties of the tether. This study also reveals that the surface immobilization-based experiments are expected to provide different kinetic and equilibrium fingerprints of the tethered ligand–receptor interactions with respect to unrestrained conditions. For example, we show that the association rate constants of ligand–receptor interactions under NT conditions are about 1 order of magnitude greater than those acquired under LT conditions. In addition, we anticipate that the nature of the linker might impact these parameters as well. Therefore, our method can be employed in biosensor technology to modulate the interaction strength of a ligand–protein complex on a sensing surface by modifying the tether length. Finally, this result has been successfully validated by using a test WDR5 mutant of unknown dissociation constant for five ST-SET1Win ligands.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.2c00621.

  • Peptide synthesis, purification, and analysis, protein expression and purification, biolayer interferometry, surface plasmon resonance, examples of BLI sensorgrams, determinations of the kinetic and equilibrium constants, scatter plots of the association rate constants versus the dissociation rate constants, examples of SPR sensorgrams, 3D plots and contour maps of the association and dissociation rate constants, supporting references (PDF)

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Author Information

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  • Corresponding Author
    • Liviu Movileanu - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States;  Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, New York 13244, United States;  The BioInspired Institute, Syracuse University, Syracuse, New York 13244, United States;  Orcidhttps://orcid.org/0000-0002-2525-3341;  Email: [email protected]
  • Authors
    • Ali Imran - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    • Brandon S. Moyer - Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States;  Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
    • Aaron J. Wolfe - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States;  Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States;  Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States;  Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
    • Michael S. Cosgrove - Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
    • Dmitrii E. Makarov - Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States;  Oden Institute for Computational Engineering and Sciences, University of Texas at Austin, Austin, Texas 78712, United States;  Orcidhttps://orcid.org/0000-0002-8421-1846
  • Author Contributions

    A.I., B.S.M., A.J.W., M.S.C., D.E.M., and L.M. designed the research. A.I., B.S.M., and D.E.M. performed research and analyzed data. A.J.W., M.S.C., and L.M. provided reagents, supervision, and funding. A.I., D.E.M., and L.M. wrote the paper.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank our colleagues in the Movileanu and Cosgrove laboratories and at Ichor Life Sciences laboratories for their comments on the manuscript and stimulating discussions as well as for their technical assistance during the very early stage of this project. This work was supported by Robert A. Welch Foundation, Grant F-1514 (to D.E.M.), the National Science Foundation, Grant CHE 1955552 (to D.E.M.), the National Cancer Institute of the U.S. National Institutes of Health, Grant R01 CA140522 (to M.S.C.), and the National Institute of General Medical Sciences of the U.S. National Institutes of Health, Grant R01 GM129429 (to L.M.).

References

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This article references 58 other publications.

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    Erlendsson, S.; Teilum, K. Binding Revisited-Avidity in Cellular Function and Signaling. Front. Mol. Biosci. 2021, 7, 615565,  DOI: 10.3389/fmolb.2020.615565
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    Reiner, A.; Isacoff, E. Y. Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy. Nat. Chem. Biol. 2014, 10, 273– 280,  DOI: 10.1038/nchembio.1458
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    Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy
    Reiner, Andreas; Isacoff, Ehud Y.
    Nature Chemical Biology (2014), 10 (4), 273-280CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to det. how the ligand occupancy controls signaling in such protein complexes. We describe a soln. to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach to study gating in ionotropic glutamate receptors (iGluRs), ligand-gated ion channels that mediate excitatory neurotransmission and plasticity at glutamatergic synapses in the brain. We probed gating in two kainate-type iGluRs, GluK2 homotetramers and GluK2-GluK5 heterotetramers. Ultrafast (submillisecond) photoswitching of an azobenzene-based ligand on specific subunits provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing. The findings have implications for signaling by locally released and spillover glutamate.
  3. 3
    Jurchenko, C.; Chang, Y.; Narui, Y.; Zhang, Y.; Salaita, K. S. Integrin-generated forces lead to streptavidin-biotin unbinding in cellular adhesions. Biophys. J. 2014, 106, 1436– 1446,  DOI: 10.1016/j.bpj.2014.01.049
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    Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions
    Jurchenko, Carol; Chang, Yuan; Narui, Yoshie; Zhang, Yun; Salaita, Khalid S.
    Biophysical Journal (2014), 106 (7), 1436-1446CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The interplay between chem. and mech. signals plays an important role in cell biol., and integrin receptors are the primary mols. involved in sensing and transducing external mech. cues. The authors used integrin-specific probes in mol. tension fluorescence microscopy to study the pN forces exerted by integrin receptors in living cells. The mol. tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, integrin receptors dissoc. streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼106 - 108 times larger than that of integrin-RGD affinity, the authors' results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, also the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estd. using traction force microscopy-based methods.
  4. 4
    Movileanu, L.; Howorka, S.; Braha, O.; Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat. Biotechnol. 2000, 18, 1091– 1095,  DOI: 10.1038/80295
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    Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore
    Movileanu, Liviu; Howorka, Stefan; Braha, Orit; Bayley, Hagan
    Nature Biotechnology (2000), 18 (10), 1091-1095CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    Here we describe a new type of biosensor element for detecting proteins in soln. at nanomolar concns. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin mol. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are obsd. as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-mol. level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resoln. compared with other kinetic measurements, such as those obtained by surface plasmon resonance.
  5. 5
    Komatsu, N.; Aoki, K.; Yamada, M.; Yukinaga, H.; Fujita, Y.; Kamioka, Y.; Matsuda, M. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 2011, 22, 4647– 4656,  DOI: 10.1091/mbc.e11-01-0072
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    Development of an optimized backbone of FRET biosensors for kinases and GTPases
    Komatsu, Naoki; Aoki, Kazuhiro; Yamada, Masashi; Yukinaga, Hiroko; Fujita, Yoshihisa; Kamioka, Yuji; Matsuda, Michiyuki
    Molecular Biology of the Cell (2011), 22 (23), 4647-4656CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    Biosensors based on the principle of Forster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling mols. Among them, intramol. FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramol. FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramol. FRET biosensors. The key concept is to exclude the "orientation-dependent" FRET and to render the biosensors completely "distance-dependent" with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramol. FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quant. evaluation of kinase inhibitors in living cells.
  6. 6
    Watkins, H. M.; Vallee-Belisle, A.; Ricci, F.; Makarov, D. E.; Plaxco, K. W. Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study. J. Am. Chem. Soc. 2012, 134, 2120– 2126,  DOI: 10.1021/ja208436p
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    Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: An experimental and theoretical study
    Watkins, Herschel M.; Vallee-Belisle, Alexis; Ricci, Francesco; Makarov, Dmitrii E.; Plaxco, Kevin W.
    Journal of the American Chemical Society (2012), 134 (4), 2120-2126CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Surface-tethered biomols. play key roles in many biol. processes and biotechnologies. However, while the phys. consequences of such surface attachment have seen significant theor. study, to date this issue has seen relatively little exptl. investigation. In response, the authors present here a quant. exptl. and theor. study of the extent to which attachment to a charged, but otherwise apparently inert, surface alters the folding free energy of a simple biomol. Specifically, the authors measured the folding free energy of a DNA stem loop both in soln. and when site-specifically attached to a neg. charged, hydroxylalkane-coated gold surface. It was found that whereas surface attachment was destabilizing at low ionic strength, it became stabilizing at ionic strengths above ∼130 mM. This behavior presumably reflects 2 competing mechanisms: excluded vol. effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the neg. charged surface. To test this hypothesis, the authors employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theor. models quant. fit the exptl. results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded vol. effects was reasonably complete and accurate.
  7. 7
    Schena, A.; Griss, R.; Johnsson, K. Modulating protein activity using tethered ligands with mutually exclusive binding sites. Nat. Commun. 2015, 6, 7830,  DOI: 10.1038/ncomms8830
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    Modulating protein activity using tethered ligands with mutually exclusive binding sites
    Schena, Alberto; Griss, Rudolf; Johnsson, Kai
    Nature Communications (2015), 6 (), 7830CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    The possibility to design proteins whose activities can be switched on and off by unrelated effector mols. would enable applications in various research areas, ranging from biosensing to synthetic biol. We describe here a general method to modulate the activity of a protein in response to the concn. of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramol. ligand-protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small mols. in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.
  8. 8
    Fahie, M. A.; Yang, B.; Pham, B.; Chen, M. Tuning the selectivity and sensitivity of an OmpG nanopore sensor by adjusting ligand tether length. ACS Sens 2016, 1, 614– 622,  DOI: 10.1021/acssensors.6b00014
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    Tuning the Selectivity and Sensitivity of an OmpG Nanopore Sensor by Adjusting Ligand Tether Length
    Fahie, Monifa A.; Yang, Bib; Pham, Bach; Chen, Min
    ACS Sensors (2016), 1 (5), 614-622CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    We have previously shown that a biotin ligand tethered to the rim of an OmpG nanopore can be used to detect biotin-binding proteins. Here, we investigate the effect of the length of the polyethylene glycol tether on the nanopore's sensitivity and selectivity. When the tether length was increased from 2 to 45 ethylene repeats, sensitivity decreased substantially for a neutral protein streptavidin and slightly for a pos. charged protein (avidin). In addn., we found that two distinct avidin binding conformations were possible when using a long tether. These conformations were sensitive to the salt concn. and applied voltage. Finally, a longer tether resulted in reduced sensitivity due to slower assocn. for a monoclonal antibiotin antibody. Our results highlight the importance of electrostatic, electroosmotic, and electrophoretic forces on nanopore binding kinetics and sensor readout.
  9. 9
    Kang, D.; Sun, S.; Kurnik, M.; Morales, D.; Dahlquist, F. W.; Plaxco, K. W. New Architecture for Reagentless, Protein-Based Electrochemical Biosensors. J. Am. Chem. Soc. 2017, 139, 12113– 12116,  DOI: 10.1021/jacs.7b05953
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    New Architecture for Reagentless, Protein-Based Electrochemical Biosensors
    Kang, Di; Sun, Sheng; Kurnik, Martin; Morales, Demosthenes; Dahlquist, Frederick W.; Plaxco, Kevin W.
    Journal of the American Chemical Society (2017), 139 (35), 12113-12116CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Here the authors demonstrate a new class of reagentless, single-step sensors for the detection of proteins and peptides that is the electrochem. analog of fluorescence polarization (fluorescence anisotropy), a versatile optical approach widely employed to this same end. The authors' electrochem. sensors consist of a redox-reporter-modified protein (the "receptor") site-specifically anchored to an electrode via a short, flexible polypeptide linker. Interaction of the receptor with its binding partner alters the efficiency with which the reporter approaches the electrode surface, thus causing a change in redox current upon voltammetric interrogation. As the first proof-of-principle the authors employed the bacterial chemotaxis protein CheY as the receptor. Interaction with either of CheY's two binding partners, the P2 domain of the chemotaxis kinase, CheA, or the 16-residue "target region" of the flagellar switch protein, FliM, leads to easily measurable changes in output current that trace Langmuir isotherms within error of those seen in soln. Phosphorylation of the electrode-bound CheY decreases its affinity for CheA-P2 and enhances its affinity for FliM in a manner likewise consistent with its behavior in soln. As expected given the proposed sensor signaling mechanism, the magnitude of the binding-induced signal change depends on the placement of the redox reporter on the receptor. Following these preliminary studies with CheY, the authors also developed and characterized addnl. sensors aimed at the detection of specific antibodies using the relevant protein antigens as the receptor. These exhibit excellent detection limits for their targets without the use of reagents or wash steps. This novel, protein-based electrochem. sensing architecture provides a new and potentially promising approach to sensors for the single-step measurement of specific proteins and peptides.
  10. 10
    Robinson-Mosher, A.; Chen, J. H.; Way, J.; Silver, P. A. Designing cell-targeted therapeutic proteins reveals the interplay between domain connectivity and cell binding. Biophys. J. 2014, 107, 2456– 2466,  DOI: 10.1016/j.bpj.2014.10.007
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    Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding
    Robinson-Mosher, Avi; Chen, Jan-Hung; Way, Jeffrey; Silver, Pamela A.
    Biophysical Journal (2014), 107 (10), 2456-2466CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question. As a result, many antibody-cytokine complexes do not achieve the desired level of cell-targeted binding and activity. Here, we address these design issues by developing a computational model to simulate the dynamics and binding kinetics of natural and engineered fusion proteins such as antibody-cytokine complexes. To verify the model, we developed a modular system in which an antibody fragment and a cytokine are conjugated via a DNA linker that allows for programmable linker geometry and protein spatial configuration. By assembling and testing several anti-CD20 antibody fragment-interferon α complexes, we showed that varying the linker length and cytokine binding affinity controlled the magnitude of cell-targeted signaling activation in a manner that agreed with the model predictions, which were expressed as dose-signaling response curves. The simulation results also revealed that there is a range of cytokine binding affinities that would achieve optimal therapeutic efficacy. This rapid prototyping platform will facilitate the rational design of antibody-cytokine complexes for improved therapeutic outcomes.
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    Nagamune, T. Biomolecular engineering for nanobio/bionanotechnology. Nano Converg. 2017, 4, 9,  DOI: 10.1186/s40580-017-0103-4
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    Biomolecular engineering for nanobio/bionanotechnology
    Nagamune Teruyuki
    Nano convergence (2017), 4 (1), 9 ISSN:2196-5404.
    Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.
  12. 12
    Kim, J. Y.; Meng, F.; Yoo, J.; Chung, H. S. Diffusion-limited association of disordered protein by non-native electrostatic interactions. Nat. Commun. 2018, 9, 4707,  DOI: 10.1038/s41467-018-06866-y
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    Diffusion-limited association of disordered protein by non-native electrostatic interactions
    Kim Jae-Yeol; Meng Fanjie; Yoo Janghyun; Chung Hoi Sung
    Nature communications (2018), 9 (1), 4707 ISSN:.
    Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes.
  13. 13
    Borgia, A.; Borgia, M. B.; Bugge, K.; Kissling, V. M.; Heidarsson, P. O.; Fernandes, C. B.; Sottini, A.; Soranno, A.; Buholzer, K. J.; Nettels, D.; Kragelund, B. B.; Best, R. B.; Schuler, B. Extreme disorder in an ultrahigh-affinity protein complex. Nature 2018, 555, 61– 66,  DOI: 10.1038/nature25762
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    Extreme disorder in an ultrahigh-affinity protein complex
    Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Petur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin
    Nature (London, United Kingdom) (2018), 555 (7694), 61-66CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Mol. communication in biol. is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiol. conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomols. Here, we demonstrated the existence of an unexpected interaction mechanism: the 2 intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α assoc. in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility, and highly dynamic character. On the basis of closely integrated expts. and mol. dynamics (MD) simulations, we showed that the interaction could be explained by the large opposite net charge of the 2 proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence anal. suggested that this interaction mechanism may be abundant in eukaryotes.
  14. 14
    Sturzenegger, F.; Zosel, F.; Holmstrom, E. D.; Buholzer, K. J.; Makarov, D. E.; Nettels, D.; Schuler, B. Transition path times of coupled folding and binding reveal the formation of an encounter complex. Nat. Commun. 2018, 9, 4708,  DOI: 10.1038/s41467-018-07043-x
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    Transition path times of coupled folding and binding reveal the formation of an encounter complex
    Sturzenegger Flurin; Zosel Franziska; Holmstrom Erik D; Buholzer Karin J; Nettels Daniel; Schuler Benjamin; Zosel Franziska; Makarov Dmitrii E; Schuler Benjamin
    Nature communications (2018), 9 (1), 4708 ISSN:.
    The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we use single-molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding. The results reveal the formation of a metastable encounter complex that is electrostatically favored and transits to the final bound state within tens of microseconds. Such measurements thus open a new window into the microscopic events governing biomolecular interactions.
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    Zosel, F.; Mercadante, D.; Nettels, D.; Schuler, B. A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction. Nat. Commun. 2018, 9, 3332,  DOI: 10.1038/s41467-018-05725-0
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    A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction
    Zosel Franziska; Mercadante Davide; Nettels Daniel; Schuler Benjamin; Zosel Franziska; Schuler Benjamin
    Nature communications (2018), 9 (1), 3332 ISSN:.
    The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while disordered, and they may fold to structured conformations on binding. Here we show that the cis/trans isomerization of peptidyl-prolyl bonds can have a pronounced effect on the interactions of IDPs. By single-molecule spectroscopy, we identify a conserved proline residue in NCBD (the nuclear-coactivator binding domain of CBP) whose cis/trans isomerization in the unbound state modulates the association and dissociation rates with its binding partner, ACTR. As a result, NCBD switches on a time scale of tens of seconds between two populations that differ in their affinities to ACTR by about an order of magnitude. Molecular dynamics simulations indicate as a cause reduced packing of the complex for the cis isomer. Peptidyl-prolyl cis/trans isomerization may be an important previously unidentified mechanism for regulating IDP interactions.
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    Mayse, L. A.; Imran, A.; Larimi, M. G.; Cosgrove, M. S.; Wolfe, A. J.; Movileanu, L. Disentangling the recognition complexity of a protein hub using a nanopore. Nat. Commun. 2022, 13, 978,  DOI: 10.1038/s41467-022-28465-8
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    Disentangling the recognition complexity of a protein hub using a nanopore
    Mayse, Lauren Ashley; Imran, Ali; Larimi, Motahareh Ghahari; Cosgrove, Michael S.; Wolfe, Aaron James; Movileanu, Liviu
    Nature Communications (2022), 13 (1), 978CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiol. and disease-like conditions, their reversible interactions with other proteins remain modestly examd. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-assocd. hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 deriv. and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.
  17. 17
    De Gennes, P.-G. Kinetics of diffusion-controlled processes in dense polymer systems. I. Nonentangled regimes. J. Chem. Phys. 1982, 76, 3316– 3321,  DOI: 10.1063/1.443328
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    Kinetics of diffusion-controlled processes in dense polymer systems. I. Nonentangled regimes
    De Gennes, P. G.
    Journal of Chemical Physics (1982), 76 (6), 3316-21CODEN: JCPSA6; ISSN:0021-9606.
    Diffusion-controlled processes were investigated where the reacting groups (A and B) are attached to long, flexible, macromols. in melts or in concd. solns. A general discussion is given of the rate consts., and 2 fundamental types of behavior are distinguished, depending on the rms displacement x(t) of one reacting group during a time t. First, if t-1x3(t) is an increasing function of time, the space vols. [∼x3(t)] explored by A and B may overlap significantly without any reaction taking place: this regime is called noncompact exploration. It is obtained in the classical case where A and B belong to small mols. and where simple diffusion prevails [x(t)∼t1/2]. This regime leads to a 2nd-order rate const. k in the chem. kinetics which is well-defined (independent of the time). In the 2nd type of behavior, if x3(t)/t is a decreasing function of time, it is called compact exploration: as soon as the space vols. explored by A and B overlap, the reaction takes place. Then the rate const. k is proportional to x3(t)/t and is thus time dependent. Here the case is analyzed of dense chains which are not long enough to be entangled (d.p. N smaller than a certain threshold Ne). At reaction times t smaller than the Rouse time TR of the chains it is shown that this is a case of compact exploration, with x(t)∼t1/4 and k(t)∼t-1/4. For t > TR, a noncompact exploration is recovered, and k∼DRR0, where DR is the Rouse diffusivity of the chains, and R0 their rms end-to-end size. Thus, k∼N-1/2 is predicted.
  18. 18
    Van Valen, D.; Haataja, M.; Phillips, R. Biochemistry on a leash: the roles of tether length and geometry in signal integration proteins. Biophys. J. 2009, 96, 1275– 1292,  DOI: 10.1016/j.bpj.2008.10.052
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    Biochemistry on a leash: The roles of tether length and geometry in signal integration proteins
    Van Valen, David; Haataja, Mikko; Phillips, Rob
    Biophysical Journal (2009), 96 (4), 1275-1292CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    We use statistical mechanics and simple ideas from polymer physics to develop a quant. model of proteins whose activity is controlled by flexibly tethered ligands and receptors. We predict how the properties of tethers influence the function of these proteins and demonstrate how their tether length dependence can be exploited to construct proteins whose integration of multiple signals can be tuned. One case study to which we apply these ideas is that of the Wiskott-Aldrich Syndrome Proteins as activators of actin polymn. More generally, tethered ligands competing with those free in soln. are common phenomena in biol., making this an important specific example of a widespread biol. idea.
  19. 19
    Ren, C. L.; Carvajal, D.; Shull, K. R.; Szleifer, I. Streptavidin-biotin binding in the presence of a polymer spacer. A theoretical description. Langmuir 2009, 25, 12283– 12292,  DOI: 10.1021/la901735d
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    Streptavidin-Biotin Binding in the Presence of a Polymer Spacer. A Theoretical Description
    Ren, Chun-lai; Carvajal, Daniel; Shull, Kenneth R.; Szleifer, Igal
    Langmuir (2009), 25 (20), 12283-12292CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The binding of streptavidin to biotin located at the terminal ends of poly(ethylene oxide) tethered to a planar surface is studied using mol. theory. The theor. model is applied to mimic expts. (Langmuir 2008, 24, 2472) performed using drop-shape anal. to study receptor-ligand binding at the oil/water interface. The authors' theor. predictions show very good agreements with the exptl. results. Furthermore, the theory enables the authors to study the thermodn. and structural behavior of the PEO-biotin + streptavidin layer. The interfacial structure, shown by the vol. fraction profiles of bound proteins and polymers, indicates that the proteins form a thick layer supported by stretched polymers, where the thickness of the layer is greater than the height of the protein. When the polymer spacer is composed of PEO (3000), a thick layer with multilayers of proteins is formed, supported by the stretched polymer chains. It was found that thick multilayers of proteins are formed when long spacers are present or at very high protein surface coverages on short spacers. This shows that the flexibility of the polymer spacer plays an important role in detg. the structure of the bound proteins due to their ability to accommodate highly distorted conformations to optimize binding and protein interactions. Protein domains are predicted when the amt. of bound proteins is small due to the existence of streptavidin-streptavidin attractive interactions. As the no. of proteins is increased, the competition between attractive interactions and steric repulsions dets. the stability and structure of the bound layer. The theory predicts that the competition between these two forces leads to a phase sepn. at higher protein concns. The point where this transition happens depends on both spacer length and protein surface coverage and is an important consideration for practical applications of these and other similar systems. If the goal is to maximize protein binding, it is favorable to be above the layer transition, as multiple layers can accommodate greater bound protein densities. If the goal is to use these bound proteins as a linker group to build more complex structures, such as when avidin or streptavidin serves as a linker between two biotinylated polymers or proteins, the optimum is to be below the layer transition such that all bound linker proteins are available for further binding.
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    Kane, R. S. Thermodynamics of multivalent interactions: influence of the linker. Langmuir 2010, 26, 8636– 8640,  DOI: 10.1021/la9047193
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    Thermodynamics of Multivalent Interactions: Influence of the Linker
    Kane, Ravi S.
    Langmuir (2010), 26 (11), 8636-8640CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    This paper describes a thermodn. anal. of multivalent interactions, with the goal of clarifying the influence of the linker on the enhancement in avidity due to multivalency. The use of multivalency represents a promising approach to inhibit undesired biol. interactions, promote desired cellular responses, and control recognition events at surfaces. Several groups have synthesized multivalent ligands that are orders of magnitude more potent than the corresponding monovalent ligands. A better understanding of the theor. basis for the large enhancements in avidity would help guide the design of more potent synthetic multivalent ligands. In particular, there has been significant controversy regarding the extent to which the loss of conformational entropy of the linker influences the enhancement in avidity due to multivalency. To help clarify this issue, we present the thermodn. anal. of a heterodivalent ligand-receptor interaction. Our anal. helps reconcile seemingly competing theor. analyses of multivalent binding. Our results indicate that the dependence of the free energy of multivalent binding on linker length can be weak even if there is a significant decrease in the conformational entropy of the linker on binding. Our results are also consistent with studies demonstrating that the use of flexible linkers represents an effective strategy to design potent multivalent ligands.
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    Reeves, D.; Cheveralls, K.; Kondev, J. Regulation of biochemical reaction rates by flexible tethers. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2011, 84, 021914,  DOI: 10.1103/PhysRevE.84.021914
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    Regulation of biochemical reaction rates by flexible tethers
    Reeves, Daniel; Cheveralls, Keith; Kondev, Jane
    Physical Review E: Statistical, Nonlinear, and Soft Matter Physics (2011), 84 (2-1), 021914/1-021914/12CODEN: PRESCM; ISSN:1539-3755. (American Physical Society)
    We explore how ligand-receptor binding kinetics can be controlled by tethering the receptor to the end of a flexible polymer. The tether confines the diffusive motion of the receptor, thus influencing the rate at which it captures ligands that are free in soln. We compute steady-state collision rates between ligand and receptor for this "tethered-capture" mechanism using a combination of analytic and numerical techniques. In doing so, we uncover a dimensionless control parameter, the "opacity," that dets. under what conditions and to what extent a tether regulates the ligand-receptor collision rate. We compute the opacity for a no. of different tethering scenarios that appear in biol. and use these results to predict the affect of changing the length and flexibility of the tether on the rate at which ligands are captured from soln.
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    Levin, M. D.; Shimizu, T. S.; Bray, D. Binding and diffusion of CheR molecules within a cluster of membrane receptors. Biophys. J. 2002, 82, 1809– 1817,  DOI: 10.1016/S0006-3495(02)75531-8
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    Binding and diffusion of CheR molecules within a cluster of membrane receptors
    Levin, Matthew D.; Shimizu, Thomas S.; Bray, Dennis
    Biophysical Journal (2002), 82 (4), 1809-1817CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Adaptation of the attractant response in Escherichia coli is attributable to the methylation of its transmembrane chemotactic receptors by the methyltransferase CheR. This protein contains two binding domains, one for the sites of methylation themselves and the other for a flexible tether at the C terminus of the receptor. We have explored the theor. consequences of this binding geometry for a CheR mol. assocd. with a cluster of chemotactic receptors. Calcns. show that the CheR mol. will bind with high net affinity to the receptor lattice, having a high probability of being attached by one or both of its domains at any instant of time. Because of the relatively low affinity of its individual domains and the close proximity of neighboring receptors, it is likely that when one domain unbinds it will reattach to the array before the other domain unbinds. Stochastic simulations show that the enzyme will move through the receptor cluster in a hand-over-hand fashion, like a gibbon swinging through the branches of a tree. We explore the possible consequences of this motion, which we term "mol. brachiation", for chemotactic adaptation and suggest that a similar mechanism may be operative in other large assemblies of protein mols.
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    Windisch, B.; Bray, D.; Duke, T. Balls and chains--a mesoscopic approach to tethered protein domains. Biophys. J. 2006, 91, 2383– 2392,  DOI: 10.1529/biophysj.105.078543
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    Balls and chains-A mesoscopic approach to tethered protein domains
    Windisch, Bernhard; Bray, Dennis; Duke, Thomas
    Biophysical Journal (2006), 91 (7), 2383-2392CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Many proteins contain regions of unstructured polypeptide chain that appear to be flexible and to undergo random thermal motion. In some cases, the unfolded sequence acts as a flexible tether that restricts the diffusion of a globular protein domain for the purpose of catalysis or self-assembly. In this article, we present a stochastic model for tethered protein domains under various conditions and solve it numerically to deduce the general and dynamic properties of these systems. A crit. domain size dependent on the length of the tether is presented, above which a spherical domain tethered to an impenetrable wall by a flexible chain displays a restricted localization between two concentric half-shells. Results suggest that the diffusion of such a spherical domain is effectively reduced in its dimensionality and able to explore the available space with high efficiency. It also becomes clear that the orientation of the ball is not independent of the distance from the tethering point but becomes more constrained as the linking tether is extended. The possible biol. significance of these and other results is discussed.
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    Shewmake, T. A.; Solis, F. J.; Gillies, R. J.; Caplan, M. R. Effects of linker length and flexibility on multivalent targeting. Biomacromolecules 2008, 9, 3057– 3064,  DOI: 10.1021/bm800529b
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    Effects of Linker Length and Flexibility on Multivalent Targeting
    Shewmake, Thomas A.; Solis, Francisco J.; Gillies, Robert J.; Caplan, Michael R.
    Biomacromolecules (2008), 9 (11), 3057-3064CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Increasing valence can enhance the ability of mol. targeting constructs to bind specifically to targeted cells for drug delivery. Here, we math. model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approx. varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce exptl. measured avidities. Also, the magnitude of VR, in conjunction with receptor d. and ligand concn., significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging.
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    Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S. Direct measurement of a tethered ligand-receptor interaction potential. Science 1997, 275, 820– 822,  DOI: 10.1126/science.275.5301.820
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    Direct measurement of a tethered ligand-receptor interaction potential
    Wong, Joyce Y.; Kuhl, Tonya L.; Israelachvili, Jacob N.; Mullah, Nasreen; Zalipsky, Samuel
    Science (Washington, D. C.) (1997), 275 (5301), 820-822CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Many biol. recognition interactions involve ligands and receptors are tethered rather than rigidly bound on a cell surface. A surface forces app. was used to directly measure the force-distance interaction between a polymer-tethered ligand and its receptor. At sepns. near the fully extended tether length, the ligands rapidly lock onto the their binding sites, pulling the ligand and receptor together. The measured interaction potential and its dynamics be modeled with std. theories of polymer and colloidal interactions.
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    Jeppesen, C.; Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S.; Marques, C. M. Impact of polymer tether length on multiple ligand-receptor bond formation. Science 2001, 293, 465– 468,  DOI: 10.1126/science.293.5529.465
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    Impact of polymer tether length on multiple ligand-receptor bond formation
    Jeppesen, Claus; Wong, Joyce Y.; Kuhl, Tonya L.; Israelachvili, Jacob N.; Mullah, Nasreen; Zalipsky, Samuel; Marques, Carlos M.
    Science (Washington, DC, United States) (2001), 293 (5529), 465-468CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The promoters of cell adhesion are ligands, which are often attached to flexible tethers that bind to surface receptors on adjacent cells. Using a combination of Monte Carlo simulations, diffusion reaction theory, and direct expts. (surface force measurements) of the biotin-streptavidin system, we have quantified polymer chain dynamics and the kinetics and spatial range of tethered ligand-receptor binding. The results show that the efficiency of strong binding does not depend solely on the mol. architecture or binding energy of the receptor-ligand pair, nor on the equil. configuration of the polymer tether, but rather on its "rare" extended conformations.
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    Leckband, D.; Israelachvili, J. Intermolecular forces in biology. Q. Rev. Biophys. 2001, 34, 105– 267,  DOI: 10.1017/S0033583501003687
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    Intermolecular forces in biology
    Leckband, Deborah; Israelachvili, Jacob
    Quarterly Reviews of Biophysics (2001), 34 (2), 105-267CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
    A review. In this review, the authors provide an overview of intermol. forces in biol. systems. They describe exptl. techniques for measuring forces between biol. mols. They discuss equil. (time-independent) interactions, and time-dependent equil. and non-equil. interactions.
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    Bauer, M.; Kékicheff, P.; Iss, J.; Fajolles, C.; Charitat, T.; Daillant, J.; Marques, C. M. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design. Nat. Commun. 2015, 6, 8117,  DOI: 10.1038/ncomms9117
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    Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design
    Bauer Martin; Kekicheff Patrick; Iss Jean; Charitat Thierry; Marques Carlos M; Bauer Martin; Fajolles Christophe; Daillant Jean
    Nature communications (2015), 6 (), 8117 ISSN:.
    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.
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    Krishnamurthy, V. M.; Semetey, V.; Bracher, P. J.; Shen, N.; Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein-ligand system. J. Am. Chem. Soc. 2007, 129, 1312– 1320,  DOI: 10.1021/ja066780e
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    Dependence of Effective Molarity on Linker Length for an Intramolecular Protein-Ligand System
    Krishnamurthy, Vijay M.; Semetey, Vincent; Bracher, Paul J.; Shen, Nan; Whitesides, George M.
    Journal of the American Chemical Society (2007), 129 (5), 1312-1320CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    This paper reports dissocn. consts. and "effective molarities" (Meff) for the intramol. binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EGn) linkers of different lengths (n = 0, 2, 5, 10, and 20) and compares these exptl. values with theor. ests. from polymer theory. As expected, the value of Meff is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker is the optimal length (n = 2) to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but only by a factor of ∼8 from n = 2 to n = 20). These exptl. results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound noncovalently to the active site of the protein, but they are quant. compatible with a model that treats the linker as a random-coil polymer. Calorimetry revealed that enthalpic interactions between the linker and the protein are not important in detg. the thermodn. of the system. Taken together, these results suggest that the manifestation of the linker in the thermodn. of binding is exclusively entropic. The values of Meff are, theor., intrinsic properties of the EGn linkers and can be used to predict the avidities of multivalent ligands with these linkers for multivalent proteins. The weak dependence of Meff on linker length suggests that multivalent ligands contg. flexible linkers that are longer than the spacing between the binding sites of a multivalent protein will be effective in binding, and that the use of flexible linkers with lengths somewhat greater than the optimal distance between binding sites is a justifiable strategy for the design of multivalent ligands.
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    Sørensen, C. S.; Kjaergaard, M. Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 23124– 23131,  DOI: 10.1073/pnas.1904813116
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    Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics
    Sorensen Charlotte S; Kjaergaard Magnus; Sorensen Charlotte S; Kjaergaard Magnus; Kjaergaard Magnus; Kjaergaard Magnus
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (46), 23124-23131 ISSN:.
    Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.
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    Patel, A.; Dharmarajan, V.; Cosgrove, M. S. Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide. J. Biol. Chem. 2008, 283, 32158– 32161,  DOI: 10.1074/jbc.C800164200
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    Structure of WDR5 Bound to Mixed Lineage Leukemia Protein-1 Peptide
    Patel, Anamika; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
    Journal of Biological Chemistry (2008), 283 (47), 32158-32161CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic disks-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine contg. motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-Å crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodn. binding expts. show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymic activity of the MLL1 core complex.
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    Song, J. J.; Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 2008, 283, 35258– 35264,  DOI: 10.1074/jbc.M806900200
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    WDR5 Interacts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket
    Song, Ji-Joon; Kingston, Robert E.
    Journal of Biological Chemistry (2008), 283 (50), 35258-35264CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.
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    Li, Y.; Han, J.; Zhang, Y.; Cao, F.; Liu, Z.; Li, S.; Wu, J.; Hu, C.; Wang, Y.; Shuai, J.; Chen, J.; Cao, L.; Li, D.; Shi, P.; Tian, C.; Zhang, J.; Dou, Y.; Li, G.; Chen, Y.; Lei, M. Structural basis for activity regulation of MLL family methyltransferases. Nature 2016, 530, 447– 452,  DOI: 10.1038/nature16952
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    Structural basis for activity regulation of MLL family methyltransferases
    Li, Yanjing; Han, Jianming; Zhang, Yuebin; Cao, Fang; Liu, Zhijun; Li, Shuai; Wu, Jian; Hu, Chunyi; Wang, Yan; Shuai, Jin; Chen, Juan; Cao, Liaoran; Li, Dangsheng; Shi, Pan; Tian, Changlin; Zhang, Jian; Dou, Yali; Li, Guohui; Chen, Yong; Lei, Ming
    Nature (London, United Kingdom) (2016), 530 (7591), 447-452CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The mixed lineage leukemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the mol. mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochem. and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.
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    Xue, H.; Yao, T.; Cao, M.; Zhu, G.; Li, Y.; Yuan, G.; Chen, Y.; Lei, M.; Huang, J. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature 2019, 573, 445– 449,  DOI: 10.1038/s41586-019-1528-1
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    Structural basis of nucleosome recognition and modification by MLL methyltransferases
    Xue, Han; Yao, Tonghui; Cao, Mi; Zhu, Guanjun; Li, Yan; Yuan, Guiyong; Chen, Yong; Lei, Ming; Huang, Jing
    Nature (London, United Kingdom) (2019), 573 (7774), 445-449CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Methyltransferases of the mixed-lineage leukemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have crit. and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are assocd. with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 Me marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favors open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules assocd. with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the assocn. between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.
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    Dharmarajan, V.; Lee, J. H.; Patel, A.; Skalnik, D. G.; Cosgrove, M. S. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases. J. Biol. Chem. 2012, 287, 27275– 27289,  DOI: 10.1074/jbc.M112.364125
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    Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases
    Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G.; Cosgrove, Michael S.
    Journal of Biological Chemistry (2012), 287 (33), 27275-27289CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are assocd. with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoppt. WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we detd. structures of WDR5 bound to six different naturally occurring Win motif sequences at resolns. ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.
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    Zhang, P.; Lee, H.; Brunzelle, J. S.; Couture, J. F. The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases. Nucleic Acids Res. 2012, 40, 4237– 4246,  DOI: 10.1093/nar/gkr1235
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    The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases
    Zhang, Pamela; Lee, Hwabin; Brunzelle, Joseph S.; Couture, Jean-Francois
    Nucleic Acids Research (2012), 40 (9), 4237-4246CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochem. and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative anal. of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.
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    Weeramange, C. J.; Fairlamb, M. S.; Singh, D.; Fenton, A. W.; Swint-Kruse, L. The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules. Protein Sci. 2020, 29, 1004,  DOI: 10.1002/pro.3827
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    Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics
    Masson, Jean-Francois
    ACS Sensors (2017), 2 (1), 16-30CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    A review. The design and application of sensors for monitoring biomols. in clin. samples is a common goal of the sensing research community. Surface plasmon resonance (SPR) and other plasmonic techniques such as localized surface plasmon resonance (LSPR) and imaging SPR are reaching a maturity level sufficient for their application in monitoring biomols. in clin. samples. In recent years, the first examples for monitoring antibodies, proteins, enzymes, drugs, small mols., peptides, and nucleic acids in biofluids collected from patients afflicted with a series of medical conditions (Alzheimer's, hepatitis, diabetes, leukemia, and cancers such as prostate and breast cancers, among others) demonstrate the progress of SPR sensing in clin. chem. This Perspective reviews the current status of the field, showcasing a series of early successes in the application of SPR for clin. anal. and detailing a series of considerations regarding sensing schemes, exposing issues with anal. in biofluids, and comparing SPR with ELISA, while providing an outlook of the challenges currently assocd. with plasmonic materials, instrumentation, microfluidics, bioreceptor selection, selection of a clin. market, and validation of a clin. assay for applying SPR sensors to clin. samples. Research opportunities are proposed to further advance the field and transition SPR biosensors from research proof-of-concept stage to actual clin. applications.
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    Pang, X.; Zhou, H. X. Rate Constants and Mechanisms of Protein-Ligand Binding. Annu. Rev. Biophys. 2017, 46, 105– 130,  DOI: 10.1146/annurev-biophys-070816-033639
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    Rate Constants and Mechanisms of Protein-Ligand Binding
    Pang, Xiaodong; Zhou, Huan-Xiang
    Annual Review of Biophysics (2017), 46 (), 105-130CODEN: ARBNCV; ISSN:1936-122X. (Annual Reviews)
    Whereas protein-ligand binding affinities have long-established prominence, binding rate consts. and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new exptl. techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate consts., can and should be quant. detd. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and phys. insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its redn. to simple rate equations; computation of binding rate consts.; quant. detn. of binding mechanisms; and elucidation of phys. factors that control binding rate consts. and mechanisms.
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    Although Intrinsically Disordered Proteins (IDPs) and Intrinsically Disordered Regions (IDRs) in folded proteins are not able to form stable structures, it is known that they play critically important roles in various biol. processes. However, despite multiple studies, the mol. mechanisms of their functioning remain not fully understood. In this work, we theor. investigate the role of IDRs in acceleration of protein-protein assocn. processes. Our hypothesis is that in protein pairs with several independent binding sites the assocn. process goes faster if some of these binding sites are located on IDRs or connected by IDRs. To test this idea, we employed anal. modeling, numerical calcns. and Brownian Dynamics computer simulations to calc. protein-protein assocn. reaction rates for ERK2-EtsΔ138 system, belonging to RAS-RAF-MEK-ERK signaling pathway in living cells. It is found that putting a binding site on IDR accelerates assocn. process by a factor of 3 to 4. Possible mol. explanations of these observations are presented, and other systems that might use this mechanism are also mentioned.
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    A review. This review focuses on recent advances in deciphering the kinetic pathway of protein complex formation, the nature of the precomplex formed through diffusion (which the authors term the "transient complex"), the transition state and other intermediates (such as the so-called encounter complex) along the assocn. pathway.
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    Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein mol. can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the "fly-casting mechanism.". We illustrate this scenario with the hypothetical kinetics of binding a single repressor mol. to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.
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    Mol. plasticity, the key to many biomol. self-assembly processes, and electrostatic steering, which guides proteins to DNA, are shown to be coupled and to facilitate DNA search. While protein flexibility is involved in induced-fit recognition and to a larger extent in intrinsically unstructured DNA binding proteins, we show that through a "tidal force" the electrostatic field of the DNA can induce flexibility and the partial unfolding of a two-state folding protein, thereby reducing its folding barrier and, thus, stimulating fly-casting. The protein binds DNA nonspecifically in a partially folded state and completes its folding when it binds the specific site. The interplay between fly-casting and electrostatics is obsd. even for weak electrostatic forces and is expected to vary with the electrostatic screening due to salt and the intrinsic folding barrier, both of which can be modulated exptl.
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    Biomol. folding and function are often coupled. During mol. recognition events, one of the binding partners may transiently or partially unfold, allowing more rapid access to a binding site. We describe a simple model for this fly-casting mechanism based on the capillarity approxn. and polymer chain statistics. The model shows that fly casting is most effective when the protein unfolding barrier is small and the part of the chain which extends toward the target is relatively rigid. These features are often seen in known examples of fly casting in protein-DNA binding. Simulations of protein-DNA binding based on well-funneled native-topol. models with electrostatic forces confirm the trends of the anal. theory.
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    Protein folding and binding are analogous processes, in which the protein 'searches' for favorable intramol. or intermol. interactions on a funnelled energy landscape. Many eukaryotic proteins are disordered under physiol. conditions, and fold into ordered structures only on binding to their cellular targets. The mechanism by which folding is coupled to binding is poorly understood, but it has been hypothesized on theor. grounds that the binding kinetics may be enhanced by a 'fly-casting' effect, where the disordered protein binds weakly and non-specifically to its target and folds as it approaches the cognate binding site. Here we show, using NMR titrns. and 15N relaxation dispersion, that the phosphorylated kinase inducible activation domain (pKID) of the transcription factor CREB forms an ensemble of transient encounter complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate to the fully bound state without dissocn. from KIX. The carboxy-terminal helix of pKID is only partially folded in the intermediate, and becomes stabilized by intermol. interactions formed in the final bound state. Future applications of our method will provide new understanding of the mol. mechanisms by which intrinsically disordered proteins perform their diverse biol. functions.
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    A review. Many cellular proteins are intrinsically disordered and undergo folding, in whole or in part, upon binding to their physiol. targets. The past few years have seen an exponential increase in papers describing characterization of intrinsically disordered proteins (IDP), both free and bound to targets. Although NMR spectroscopy remains the favored tool, a no. of new biophys. techniques are proving exceptionally useful in defining the limits of the conformational ensembles. Advances have been made in prediction of the recognition elements in IDP, in elucidating the kinetics and mechanism of the coupled folding and binding process, and in understanding the role of post-translational modifications in tuning the biol. response. Here, the authors review these and other recent advances that are providing new insights into the conformational propensities and interactions of IDP and are beginning to reveal general principles underlying their biol. functions.
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    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (13), 4874-4879CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-mol. Foerster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concn., but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the obsd. behavior can be explained quant. if the polymeric nature of both the IDPs and the crowding mols. is taken into account explicitly. Our results suggest that excluded vol. interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.
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    Mollica, L.; Bessa, L. M.; Hanoulle, X.; Jensen, M. R.; Blackledge, M.; Schneider, R. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment. Front. Mol. Biosci. 2016, 3, 52,  DOI: 10.3389/fmolb.2016.00052
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    A review. In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an assocd. function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiol. partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the "fly-casting" hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Exptl., these questions remain difficult to address. Here, we review exptl. studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theor. concepts and numerical methods that can be applied to describe these interactions at the at. level. The available literature suggests that the kinetic and thermodn. parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes obsd. exptl. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.
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    The intrinsically disordered protein (IDP) has distinct properties both phys. and biol.: it often becomes folded when binding to the target and is frequently involved in signal transduction. The phys. property seems to be compatible with the biol. property where fast assocn. and dissocn. between IDP and the target are required. While fast assocn. has been well studied, fueled by the fly-casting mechanism, the dissocn. kinetics has received less attention. Here, the authors studied how intrinsic disorder affects the dissocn. kinetics, as well as the assocn. kinetics, paying attention to the interaction strength at the binding site (i.e., the quality of the "fly lure"). Coarse-grained mol. dynamics simulation of the pKID-KIX system (pKID is an intrinsically disordered region of CREB protein and KIX is the KID-interaction domain od CREB-binding protein), a well-studied IDP system, shows that the assocn. rate becomes larger as the disorder-inducing flexibility that was imparted to the model is increased, but the acceleration is marginal and turns into deceleration as the quality of the fly lure is worsened. In contrast, the dissocn. rate is greatly enhanced as the disorder is increased, indicating that intrinsic disorder serves for rapid signal switching more effectively through dissocn. than assocn.
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    Huang, Y.; Liu, Z. Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the “fly-casting” mechanism. J. Mol. Biol. 2009, 393, 1143– 1159,  DOI: 10.1016/j.jmb.2009.09.010
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    Kinetic Advantage of Intrinsically Disordered Proteins in Coupled Folding-Binding Process: A Critical Assessment of the "Fly-Casting" Mechanism
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    Intrinsically disordered proteins (IDPs) are recognized to play important roles in many biol. functions such as transcriptional and translational regulation, cellular signal transduction, protein phosphorylation, and mol. assemblies. The coupling of folding with binding through a "fly-casting" mechanism has been proposed to account for the fast binding kinetics of IDPs. In this article, exptl. data from the literature were collated to verify the kinetic advantages of IDPs, while mol. simulations were performed to clarify the origin of the kinetic advantages. The phosphorylated KID-kinase-inducible domain interacting domain (KIX) complex was used as an example in the simulations. By modifying a coarse-grained model with a native-centric Go-like potential, we were able to continuously tune the degree of disorder of the phosphorylated KID domain and thus investigate the intrinsic role of chain flexibility in binding kinetics. The simulations show that the "fly-casting" effect is not only due to the greater capture radii of IDPs. The coupling of folding with binding of IDPs leads to a significant redn. in binding free-energy barrier. Such a redn. accelerates the binding process. Although the greater capture radius has been regarded as the main factor in promoting the binding rate of IDPs, we found that this parameter will also lead to the slower translational diffusion of IDPs when compared with ordered proteins. As a result, the capture rate of IDPs was found to be slower than that of ordered proteins. The main origin of the faster binding for IDPs are the fewer encounter times required before the formation of the final binding complex. The roles of the interchain native contacts fraction (Qb) and the mass-center distance (ΔR) as reaction coordinates are also discussed.
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    We give a theor. anal. of bead motion in tethered-particle expts., a single-mol. technique that has been used to explore the dynamics of a variety of macromols. of biol. interest. Our anal. reveals that the proximity of the tethered bead to a nearby surface gives rise to a vol.-exclusion effect, resulting in an entropic stretching-force on the mol. that changes its statistical properties. In addn., vol. exclusion brings about intriguing scaling relations between key observables (statistical moments of the bead) and parameters such as bead size and contour length of the mol. We present analytic and numerical results for these effects in both flexible and semiflexible tethers. Finally, our results give a precise, exptl. testable prediction for the probability distribution of the bead center measured from the polymer attachment point.
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    Perspective: Mechanochemistry of biological and synthetic molecules
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    Coupling of mech. forces and chem. transformations is central to the biophysics of mol. machines, polymer chem., fracture mechanics, tribol., and other disciplines. As a consequence, the same phys. principles and theor. models should be applicable in all of those fields; in fact, similar models have been invoked (and often repeatedly reinvented) to describe, for example, cell adhesion, dry and wet friction, propagation of cracks, and action of mol. motors. This perspective offers a unified view of these phenomena, described in terms of chem. kinetics with rates of elementary steps that are force dependent. The central question is then to describe how the rate of a chem. transformation (and its other measurable properties such as the transition path) depends on the applied force. I will describe phys. models used to answer this question and compare them with exptl. measurements, which employ single-mol. force spectroscopy and which become increasingly common. Multidimensionality of the underlying mol. energy landscapes and the ensuing frequent misalignment between chem. and mech. coordinates result in a no. of distinct scenarios, each showing a nontrivial force dependence of the reaction rate. I will discuss these scenarios, their commonness (or its lack), and the prospects for their exptl. validation. Finally, I will discuss open issues in the field. (c) 2016 American Institute of Physics.
  58. 58
    Imran, A.; Moyer, B. S.; Canning, A. J.; Kalina, D.; Duncan, T. M.; Moody, K. J.; Wolfe, A. J.; Cosgrove, M. S.; Movileanu, L. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions. Biochem. J. 2021, 478, 2145– 2161,  DOI: 10.1042/BCJ20210253
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    Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions
    Imran, Ali; Moyer, Brandon S.; Canning, Ashley J.; Kalina, Dan; Duncan, Thomas M.; Moody, Kelsey J.; Wolfe, Aaron J.; Cosgrove, Michael S.; Movileanu, Liviu
    Biochemical Journal (2021), 478 (11), 2145-2161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Recent advances in quant. proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their phys. assocns. with other proteins are ambiguous. A quant. understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient assocns. between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow assocn. kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissocn. kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnol.

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  • Abstract

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    Figure 1

    Figure 1. WDR5 protein interacting with the SET1Win peptide ligands under different conditions. WDR5 is shown in orange, while SET1Win ligands are shown in magenta. Bound interacting partners are shown in blue. Lightly colored receptors and ligands indicate interacting partners in the background. (a) Biotinylated ST-SET1Win ligands were chemically attached onto a streptavidin-coated biolayer interferometry (BLI) sensor surface. Either WDR5 proteins or one of its mutants was freely movable in solution. (b) The same system as in (a), but with LT-SET1Win ligands. (c) Either WDR5 proteins or one of its mutants was immobilized onto a surface plasmon resonance (SPR) chip surface, whereas the NT-SET1Win ligands were freely movable in solution.

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    Figure 2

    Figure 2. Scatter plots of kinetic and equilibrium constants for ST-SET1Win and LT-SET1Win ligands. (a) Association rate constants ka-ST of ST-SET1Win-WDR5 complexes plotted against association rate constants ka-LT of LT-SET1Win-WDR5 complexes. Points above the blue line correspond to complexes with faster association rate constants for ST-SET1Win ligands, while points below correspond to interactions with slower association rate constants for ST-SET1Win ligands. (b) Dissociation rate constants kd-ST of ST-SET1Win–WDR5 complexes plotted against dissociation rate constants kd-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to complexes with faster dissociation rate constants for ST-SET1Win ligands. (c) Equilibrium dissociation constants KD-ST of ST-SET1Win–WDR5 complexes plotted against equilibrium dissociation constants KD-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to less stable complexes with ST-SET1Win ligands. m indicates the slopes of linear fits in (b) and (c). Data represent mean ± s.d. which resulted from three independent BLI sensorgrams.

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    Figure 3

    Figure 3. Scatter plots of kinetic and equilibrium constants for NT-SET1Win and LT-SET1Win ligands. (a) Association rate constants ka-NT of NT-SET1Win–WDR5 complexes plotted against association rate constants ka-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to interactions with faster association rate constants for NT-SET1Win ligands. (b) Dissociation rate constants kd-NT of NT-SET1Win–WDR5 complexes plotted against dissociation rate constants kd-LT of LT-SET1Win–WDR5 complexes. Points above the blue line correspond to interactions with faster dissociation rate constants for NT-SET1Win ligands. (c) Equilibrium dissociation constants KD-NT values of NT-SET1Win–WDR5 complexes plotted against equilibrium dissociation constants KD-LT of LT-SET1Win–WDR5 complexes. Points below the blue line correspond to more stable complexes with LT-SET1Win ligands. m indicates the slopes of linear fits in all panels. Data represent mean ± s.d. which resulted from three independent BLI sensorgrams.

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    Figure 4

    Figure 4. 3D plots and contour maps of normalized KD constants. (a) Qualitative free energy landscapes of SET1Win–WDR5 interactions when NT-SET1Win (NT), ST-SET1Win (ST), and LT-SET1Win (LT) peptide ligands were used. Vertical lines 1, 2, and 3, which are marked in cyan, indicate the differential free energy barriers due to unrestrained diffusion of the ligand, fly casting mechanism, and repulsion entropic forces of the receptor from the sensor surface, respectively. (b) Bar graph and (c) contour map of KD-ST values for the interaction of ST-SET1Win ligands, with WDR5 and its mutants, divided by their corresponding KD-NT values measured with the corresponding NT-SET1Win ligands. (d) Bar graph and (e) contour map of KD-LT values for the interaction of LT-SET1Win ligands, with WDR5 and its mutants, divided by their corresponding KD-NT values measured with the corresponding NT-SET1Win ligands. KD-ST and KD-LT for MLL4Win–F133L interactions could not be quantitatively determined by using BLI measurements. These data points are colored in black.

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      The possibility to design proteins whose activities can be switched on and off by unrelated effector mols. would enable applications in various research areas, ranging from biosensing to synthetic biol. We describe here a general method to modulate the activity of a protein in response to the concn. of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramol. ligand-protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small mols. in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.
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      We have previously shown that a biotin ligand tethered to the rim of an OmpG nanopore can be used to detect biotin-binding proteins. Here, we investigate the effect of the length of the polyethylene glycol tether on the nanopore's sensitivity and selectivity. When the tether length was increased from 2 to 45 ethylene repeats, sensitivity decreased substantially for a neutral protein streptavidin and slightly for a pos. charged protein (avidin). In addn., we found that two distinct avidin binding conformations were possible when using a long tether. These conformations were sensitive to the salt concn. and applied voltage. Finally, a longer tether resulted in reduced sensitivity due to slower assocn. for a monoclonal antibiotin antibody. Our results highlight the importance of electrostatic, electroosmotic, and electrophoretic forces on nanopore binding kinetics and sensor readout.
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      The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question. As a result, many antibody-cytokine complexes do not achieve the desired level of cell-targeted binding and activity. Here, we address these design issues by developing a computational model to simulate the dynamics and binding kinetics of natural and engineered fusion proteins such as antibody-cytokine complexes. To verify the model, we developed a modular system in which an antibody fragment and a cytokine are conjugated via a DNA linker that allows for programmable linker geometry and protein spatial configuration. By assembling and testing several anti-CD20 antibody fragment-interferon α complexes, we showed that varying the linker length and cytokine binding affinity controlled the magnitude of cell-targeted signaling activation in a manner that agreed with the model predictions, which were expressed as dose-signaling response curves. The simulation results also revealed that there is a range of cytokine binding affinities that would achieve optimal therapeutic efficacy. This rapid prototyping platform will facilitate the rational design of antibody-cytokine complexes for improved therapeutic outcomes.
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      Biomolecular engineering for nanobio/bionanotechnology
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      Nano convergence (2017), 4 (1), 9 ISSN:2196-5404.
      Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.
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      Diffusion-limited association of disordered protein by non-native electrostatic interactions
      Kim Jae-Yeol; Meng Fanjie; Yoo Janghyun; Chung Hoi Sung
      Nature communications (2018), 9 (1), 4707 ISSN:.
      Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes.
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      Borgia, A.; Borgia, M. B.; Bugge, K.; Kissling, V. M.; Heidarsson, P. O.; Fernandes, C. B.; Sottini, A.; Soranno, A.; Buholzer, K. J.; Nettels, D.; Kragelund, B. B.; Best, R. B.; Schuler, B. Extreme disorder in an ultrahigh-affinity protein complex. Nature 2018, 555, 61– 66,  DOI: 10.1038/nature25762
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      Extreme disorder in an ultrahigh-affinity protein complex
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      Mol. communication in biol. is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiol. conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomols. Here, we demonstrated the existence of an unexpected interaction mechanism: the 2 intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α assoc. in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility, and highly dynamic character. On the basis of closely integrated expts. and mol. dynamics (MD) simulations, we showed that the interaction could be explained by the large opposite net charge of the 2 proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence anal. suggested that this interaction mechanism may be abundant in eukaryotes.
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      Sturzenegger, F.; Zosel, F.; Holmstrom, E. D.; Buholzer, K. J.; Makarov, D. E.; Nettels, D.; Schuler, B. Transition path times of coupled folding and binding reveal the formation of an encounter complex. Nat. Commun. 2018, 9, 4708,  DOI: 10.1038/s41467-018-07043-x
      14
      Transition path times of coupled folding and binding reveal the formation of an encounter complex
      Sturzenegger Flurin; Zosel Franziska; Holmstrom Erik D; Buholzer Karin J; Nettels Daniel; Schuler Benjamin; Zosel Franziska; Makarov Dmitrii E; Schuler Benjamin
      Nature communications (2018), 9 (1), 4708 ISSN:.
      The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we use single-molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding. The results reveal the formation of a metastable encounter complex that is electrostatically favored and transits to the final bound state within tens of microseconds. Such measurements thus open a new window into the microscopic events governing biomolecular interactions.
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      A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction
      Zosel Franziska; Mercadante Davide; Nettels Daniel; Schuler Benjamin; Zosel Franziska; Schuler Benjamin
      Nature communications (2018), 9 (1), 3332 ISSN:.
      The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while disordered, and they may fold to structured conformations on binding. Here we show that the cis/trans isomerization of peptidyl-prolyl bonds can have a pronounced effect on the interactions of IDPs. By single-molecule spectroscopy, we identify a conserved proline residue in NCBD (the nuclear-coactivator binding domain of CBP) whose cis/trans isomerization in the unbound state modulates the association and dissociation rates with its binding partner, ACTR. As a result, NCBD switches on a time scale of tens of seconds between two populations that differ in their affinities to ACTR by about an order of magnitude. Molecular dynamics simulations indicate as a cause reduced packing of the complex for the cis isomer. Peptidyl-prolyl cis/trans isomerization may be an important previously unidentified mechanism for regulating IDP interactions.
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      Mayse, L. A.; Imran, A.; Larimi, M. G.; Cosgrove, M. S.; Wolfe, A. J.; Movileanu, L. Disentangling the recognition complexity of a protein hub using a nanopore. Nat. Commun. 2022, 13, 978,  DOI: 10.1038/s41467-022-28465-8
      16
      Disentangling the recognition complexity of a protein hub using a nanopore
      Mayse, Lauren Ashley; Imran, Ali; Larimi, Motahareh Ghahari; Cosgrove, Michael S.; Wolfe, Aaron James; Movileanu, Liviu
      Nature Communications (2022), 13 (1), 978CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiol. and disease-like conditions, their reversible interactions with other proteins remain modestly examd. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-assocd. hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 deriv. and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.
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      De Gennes, P.-G. Kinetics of diffusion-controlled processes in dense polymer systems. I. Nonentangled regimes. J. Chem. Phys. 1982, 76, 3316– 3321,  DOI: 10.1063/1.443328
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      Kinetics of diffusion-controlled processes in dense polymer systems. I. Nonentangled regimes
      De Gennes, P. G.
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      Diffusion-controlled processes were investigated where the reacting groups (A and B) are attached to long, flexible, macromols. in melts or in concd. solns. A general discussion is given of the rate consts., and 2 fundamental types of behavior are distinguished, depending on the rms displacement x(t) of one reacting group during a time t. First, if t-1x3(t) is an increasing function of time, the space vols. [∼x3(t)] explored by A and B may overlap significantly without any reaction taking place: this regime is called noncompact exploration. It is obtained in the classical case where A and B belong to small mols. and where simple diffusion prevails [x(t)∼t1/2]. This regime leads to a 2nd-order rate const. k in the chem. kinetics which is well-defined (independent of the time). In the 2nd type of behavior, if x3(t)/t is a decreasing function of time, it is called compact exploration: as soon as the space vols. explored by A and B overlap, the reaction takes place. Then the rate const. k is proportional to x3(t)/t and is thus time dependent. Here the case is analyzed of dense chains which are not long enough to be entangled (d.p. N smaller than a certain threshold Ne). At reaction times t smaller than the Rouse time TR of the chains it is shown that this is a case of compact exploration, with x(t)∼t1/4 and k(t)∼t-1/4. For t > TR, a noncompact exploration is recovered, and k∼DRR0, where DR is the Rouse diffusivity of the chains, and R0 their rms end-to-end size. Thus, k∼N-1/2 is predicted.
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      Van Valen, D.; Haataja, M.; Phillips, R. Biochemistry on a leash: the roles of tether length and geometry in signal integration proteins. Biophys. J. 2009, 96, 1275– 1292,  DOI: 10.1016/j.bpj.2008.10.052
      18
      Biochemistry on a leash: The roles of tether length and geometry in signal integration proteins
      Van Valen, David; Haataja, Mikko; Phillips, Rob
      Biophysical Journal (2009), 96 (4), 1275-1292CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      We use statistical mechanics and simple ideas from polymer physics to develop a quant. model of proteins whose activity is controlled by flexibly tethered ligands and receptors. We predict how the properties of tethers influence the function of these proteins and demonstrate how their tether length dependence can be exploited to construct proteins whose integration of multiple signals can be tuned. One case study to which we apply these ideas is that of the Wiskott-Aldrich Syndrome Proteins as activators of actin polymn. More generally, tethered ligands competing with those free in soln. are common phenomena in biol., making this an important specific example of a widespread biol. idea.
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      Ren, C. L.; Carvajal, D.; Shull, K. R.; Szleifer, I. Streptavidin-biotin binding in the presence of a polymer spacer. A theoretical description. Langmuir 2009, 25, 12283– 12292,  DOI: 10.1021/la901735d
      19
      Streptavidin-Biotin Binding in the Presence of a Polymer Spacer. A Theoretical Description
      Ren, Chun-lai; Carvajal, Daniel; Shull, Kenneth R.; Szleifer, Igal
      Langmuir (2009), 25 (20), 12283-12292CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The binding of streptavidin to biotin located at the terminal ends of poly(ethylene oxide) tethered to a planar surface is studied using mol. theory. The theor. model is applied to mimic expts. (Langmuir 2008, 24, 2472) performed using drop-shape anal. to study receptor-ligand binding at the oil/water interface. The authors' theor. predictions show very good agreements with the exptl. results. Furthermore, the theory enables the authors to study the thermodn. and structural behavior of the PEO-biotin + streptavidin layer. The interfacial structure, shown by the vol. fraction profiles of bound proteins and polymers, indicates that the proteins form a thick layer supported by stretched polymers, where the thickness of the layer is greater than the height of the protein. When the polymer spacer is composed of PEO (3000), a thick layer with multilayers of proteins is formed, supported by the stretched polymer chains. It was found that thick multilayers of proteins are formed when long spacers are present or at very high protein surface coverages on short spacers. This shows that the flexibility of the polymer spacer plays an important role in detg. the structure of the bound proteins due to their ability to accommodate highly distorted conformations to optimize binding and protein interactions. Protein domains are predicted when the amt. of bound proteins is small due to the existence of streptavidin-streptavidin attractive interactions. As the no. of proteins is increased, the competition between attractive interactions and steric repulsions dets. the stability and structure of the bound layer. The theory predicts that the competition between these two forces leads to a phase sepn. at higher protein concns. The point where this transition happens depends on both spacer length and protein surface coverage and is an important consideration for practical applications of these and other similar systems. If the goal is to maximize protein binding, it is favorable to be above the layer transition, as multiple layers can accommodate greater bound protein densities. If the goal is to use these bound proteins as a linker group to build more complex structures, such as when avidin or streptavidin serves as a linker between two biotinylated polymers or proteins, the optimum is to be below the layer transition such that all bound linker proteins are available for further binding.
    20. 20
      Kane, R. S. Thermodynamics of multivalent interactions: influence of the linker. Langmuir 2010, 26, 8636– 8640,  DOI: 10.1021/la9047193
      20
      Thermodynamics of Multivalent Interactions: Influence of the Linker
      Kane, Ravi S.
      Langmuir (2010), 26 (11), 8636-8640CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      This paper describes a thermodn. anal. of multivalent interactions, with the goal of clarifying the influence of the linker on the enhancement in avidity due to multivalency. The use of multivalency represents a promising approach to inhibit undesired biol. interactions, promote desired cellular responses, and control recognition events at surfaces. Several groups have synthesized multivalent ligands that are orders of magnitude more potent than the corresponding monovalent ligands. A better understanding of the theor. basis for the large enhancements in avidity would help guide the design of more potent synthetic multivalent ligands. In particular, there has been significant controversy regarding the extent to which the loss of conformational entropy of the linker influences the enhancement in avidity due to multivalency. To help clarify this issue, we present the thermodn. anal. of a heterodivalent ligand-receptor interaction. Our anal. helps reconcile seemingly competing theor. analyses of multivalent binding. Our results indicate that the dependence of the free energy of multivalent binding on linker length can be weak even if there is a significant decrease in the conformational entropy of the linker on binding. Our results are also consistent with studies demonstrating that the use of flexible linkers represents an effective strategy to design potent multivalent ligands.
    21. 21
      Reeves, D.; Cheveralls, K.; Kondev, J. Regulation of biochemical reaction rates by flexible tethers. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2011, 84, 021914,  DOI: 10.1103/PhysRevE.84.021914
      21
      Regulation of biochemical reaction rates by flexible tethers
      Reeves, Daniel; Cheveralls, Keith; Kondev, Jane
      Physical Review E: Statistical, Nonlinear, and Soft Matter Physics (2011), 84 (2-1), 021914/1-021914/12CODEN: PRESCM; ISSN:1539-3755. (American Physical Society)
      We explore how ligand-receptor binding kinetics can be controlled by tethering the receptor to the end of a flexible polymer. The tether confines the diffusive motion of the receptor, thus influencing the rate at which it captures ligands that are free in soln. We compute steady-state collision rates between ligand and receptor for this "tethered-capture" mechanism using a combination of analytic and numerical techniques. In doing so, we uncover a dimensionless control parameter, the "opacity," that dets. under what conditions and to what extent a tether regulates the ligand-receptor collision rate. We compute the opacity for a no. of different tethering scenarios that appear in biol. and use these results to predict the affect of changing the length and flexibility of the tether on the rate at which ligands are captured from soln.
    22. 22
      Levin, M. D.; Shimizu, T. S.; Bray, D. Binding and diffusion of CheR molecules within a cluster of membrane receptors. Biophys. J. 2002, 82, 1809– 1817,  DOI: 10.1016/S0006-3495(02)75531-8
      22
      Binding and diffusion of CheR molecules within a cluster of membrane receptors
      Levin, Matthew D.; Shimizu, Thomas S.; Bray, Dennis
      Biophysical Journal (2002), 82 (4), 1809-1817CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Adaptation of the attractant response in Escherichia coli is attributable to the methylation of its transmembrane chemotactic receptors by the methyltransferase CheR. This protein contains two binding domains, one for the sites of methylation themselves and the other for a flexible tether at the C terminus of the receptor. We have explored the theor. consequences of this binding geometry for a CheR mol. assocd. with a cluster of chemotactic receptors. Calcns. show that the CheR mol. will bind with high net affinity to the receptor lattice, having a high probability of being attached by one or both of its domains at any instant of time. Because of the relatively low affinity of its individual domains and the close proximity of neighboring receptors, it is likely that when one domain unbinds it will reattach to the array before the other domain unbinds. Stochastic simulations show that the enzyme will move through the receptor cluster in a hand-over-hand fashion, like a gibbon swinging through the branches of a tree. We explore the possible consequences of this motion, which we term "mol. brachiation", for chemotactic adaptation and suggest that a similar mechanism may be operative in other large assemblies of protein mols.
    23. 23
      Windisch, B.; Bray, D.; Duke, T. Balls and chains--a mesoscopic approach to tethered protein domains. Biophys. J. 2006, 91, 2383– 2392,  DOI: 10.1529/biophysj.105.078543
      23
      Balls and chains-A mesoscopic approach to tethered protein domains
      Windisch, Bernhard; Bray, Dennis; Duke, Thomas
      Biophysical Journal (2006), 91 (7), 2383-2392CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Many proteins contain regions of unstructured polypeptide chain that appear to be flexible and to undergo random thermal motion. In some cases, the unfolded sequence acts as a flexible tether that restricts the diffusion of a globular protein domain for the purpose of catalysis or self-assembly. In this article, we present a stochastic model for tethered protein domains under various conditions and solve it numerically to deduce the general and dynamic properties of these systems. A crit. domain size dependent on the length of the tether is presented, above which a spherical domain tethered to an impenetrable wall by a flexible chain displays a restricted localization between two concentric half-shells. Results suggest that the diffusion of such a spherical domain is effectively reduced in its dimensionality and able to explore the available space with high efficiency. It also becomes clear that the orientation of the ball is not independent of the distance from the tethering point but becomes more constrained as the linking tether is extended. The possible biol. significance of these and other results is discussed.
    24. 24
      Shewmake, T. A.; Solis, F. J.; Gillies, R. J.; Caplan, M. R. Effects of linker length and flexibility on multivalent targeting. Biomacromolecules 2008, 9, 3057– 3064,  DOI: 10.1021/bm800529b
      24
      Effects of Linker Length and Flexibility on Multivalent Targeting
      Shewmake, Thomas A.; Solis, Francisco J.; Gillies, Robert J.; Caplan, Michael R.
      Biomacromolecules (2008), 9 (11), 3057-3064CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Increasing valence can enhance the ability of mol. targeting constructs to bind specifically to targeted cells for drug delivery. Here, we math. model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approx. varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce exptl. measured avidities. Also, the magnitude of VR, in conjunction with receptor d. and ligand concn., significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging.
    25. 25
      Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S. Direct measurement of a tethered ligand-receptor interaction potential. Science 1997, 275, 820– 822,  DOI: 10.1126/science.275.5301.820
      25
      Direct measurement of a tethered ligand-receptor interaction potential
      Wong, Joyce Y.; Kuhl, Tonya L.; Israelachvili, Jacob N.; Mullah, Nasreen; Zalipsky, Samuel
      Science (Washington, D. C.) (1997), 275 (5301), 820-822CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Many biol. recognition interactions involve ligands and receptors are tethered rather than rigidly bound on a cell surface. A surface forces app. was used to directly measure the force-distance interaction between a polymer-tethered ligand and its receptor. At sepns. near the fully extended tether length, the ligands rapidly lock onto the their binding sites, pulling the ligand and receptor together. The measured interaction potential and its dynamics be modeled with std. theories of polymer and colloidal interactions.
    26. 26
      Jeppesen, C.; Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S.; Marques, C. M. Impact of polymer tether length on multiple ligand-receptor bond formation. Science 2001, 293, 465– 468,  DOI: 10.1126/science.293.5529.465
      26
      Impact of polymer tether length on multiple ligand-receptor bond formation
      Jeppesen, Claus; Wong, Joyce Y.; Kuhl, Tonya L.; Israelachvili, Jacob N.; Mullah, Nasreen; Zalipsky, Samuel; Marques, Carlos M.
      Science (Washington, DC, United States) (2001), 293 (5529), 465-468CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The promoters of cell adhesion are ligands, which are often attached to flexible tethers that bind to surface receptors on adjacent cells. Using a combination of Monte Carlo simulations, diffusion reaction theory, and direct expts. (surface force measurements) of the biotin-streptavidin system, we have quantified polymer chain dynamics and the kinetics and spatial range of tethered ligand-receptor binding. The results show that the efficiency of strong binding does not depend solely on the mol. architecture or binding energy of the receptor-ligand pair, nor on the equil. configuration of the polymer tether, but rather on its "rare" extended conformations.
    27. 27
      Leckband, D.; Israelachvili, J. Intermolecular forces in biology. Q. Rev. Biophys. 2001, 34, 105– 267,  DOI: 10.1017/S0033583501003687
      27
      Intermolecular forces in biology
      Leckband, Deborah; Israelachvili, Jacob
      Quarterly Reviews of Biophysics (2001), 34 (2), 105-267CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
      A review. In this review, the authors provide an overview of intermol. forces in biol. systems. They describe exptl. techniques for measuring forces between biol. mols. They discuss equil. (time-independent) interactions, and time-dependent equil. and non-equil. interactions.
    28. 28
      Bauer, M.; Kékicheff, P.; Iss, J.; Fajolles, C.; Charitat, T.; Daillant, J.; Marques, C. M. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design. Nat. Commun. 2015, 6, 8117,  DOI: 10.1038/ncomms9117
      28
      Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design
      Bauer Martin; Kekicheff Patrick; Iss Jean; Charitat Thierry; Marques Carlos M; Bauer Martin; Fajolles Christophe; Daillant Jean
      Nature communications (2015), 6 (), 8117 ISSN:.
      Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.
    29. 29
      Krishnamurthy, V. M.; Semetey, V.; Bracher, P. J.; Shen, N.; Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein-ligand system. J. Am. Chem. Soc. 2007, 129, 1312– 1320,  DOI: 10.1021/ja066780e
      29
      Dependence of Effective Molarity on Linker Length for an Intramolecular Protein-Ligand System
      Krishnamurthy, Vijay M.; Semetey, Vincent; Bracher, Paul J.; Shen, Nan; Whitesides, George M.
      Journal of the American Chemical Society (2007), 129 (5), 1312-1320CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This paper reports dissocn. consts. and "effective molarities" (Meff) for the intramol. binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EGn) linkers of different lengths (n = 0, 2, 5, 10, and 20) and compares these exptl. values with theor. ests. from polymer theory. As expected, the value of Meff is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker is the optimal length (n = 2) to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but only by a factor of ∼8 from n = 2 to n = 20). These exptl. results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound noncovalently to the active site of the protein, but they are quant. compatible with a model that treats the linker as a random-coil polymer. Calorimetry revealed that enthalpic interactions between the linker and the protein are not important in detg. the thermodn. of the system. Taken together, these results suggest that the manifestation of the linker in the thermodn. of binding is exclusively entropic. The values of Meff are, theor., intrinsic properties of the EGn linkers and can be used to predict the avidities of multivalent ligands with these linkers for multivalent proteins. The weak dependence of Meff on linker length suggests that multivalent ligands contg. flexible linkers that are longer than the spacing between the binding sites of a multivalent protein will be effective in binding, and that the use of flexible linkers with lengths somewhat greater than the optimal distance between binding sites is a justifiable strategy for the design of multivalent ligands.
    30. 30
      Sørensen, C. S.; Kjaergaard, M. Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 23124– 23131,  DOI: 10.1073/pnas.1904813116
      30
      Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics
      Sorensen Charlotte S; Kjaergaard Magnus; Sorensen Charlotte S; Kjaergaard Magnus; Kjaergaard Magnus; Kjaergaard Magnus
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (46), 23124-23131 ISSN:.
      Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.
    31. 31
      Patel, A.; Dharmarajan, V.; Cosgrove, M. S. Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide. J. Biol. Chem. 2008, 283, 32158– 32161,  DOI: 10.1074/jbc.C800164200
      31
      Structure of WDR5 Bound to Mixed Lineage Leukemia Protein-1 Peptide
      Patel, Anamika; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
      Journal of Biological Chemistry (2008), 283 (47), 32158-32161CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic disks-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine contg. motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-Å crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodn. binding expts. show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymic activity of the MLL1 core complex.
    32. 32
      Song, J. J.; Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 2008, 283, 35258– 35264,  DOI: 10.1074/jbc.M806900200
      32
      WDR5 Interacts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket
      Song, Ji-Joon; Kingston, Robert E.
      Journal of Biological Chemistry (2008), 283 (50), 35258-35264CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.
    33. 33
      Li, Y.; Han, J.; Zhang, Y.; Cao, F.; Liu, Z.; Li, S.; Wu, J.; Hu, C.; Wang, Y.; Shuai, J.; Chen, J.; Cao, L.; Li, D.; Shi, P.; Tian, C.; Zhang, J.; Dou, Y.; Li, G.; Chen, Y.; Lei, M. Structural basis for activity regulation of MLL family methyltransferases. Nature 2016, 530, 447– 452,  DOI: 10.1038/nature16952
      33
      Structural basis for activity regulation of MLL family methyltransferases
      Li, Yanjing; Han, Jianming; Zhang, Yuebin; Cao, Fang; Liu, Zhijun; Li, Shuai; Wu, Jian; Hu, Chunyi; Wang, Yan; Shuai, Jin; Chen, Juan; Cao, Liaoran; Li, Dangsheng; Shi, Pan; Tian, Changlin; Zhang, Jian; Dou, Yali; Li, Guohui; Chen, Yong; Lei, Ming
      Nature (London, United Kingdom) (2016), 530 (7591), 447-452CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The mixed lineage leukemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the mol. mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochem. and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.
    34. 34
      Xue, H.; Yao, T.; Cao, M.; Zhu, G.; Li, Y.; Yuan, G.; Chen, Y.; Lei, M.; Huang, J. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature 2019, 573, 445– 449,  DOI: 10.1038/s41586-019-1528-1
      34
      Structural basis of nucleosome recognition and modification by MLL methyltransferases
      Xue, Han; Yao, Tonghui; Cao, Mi; Zhu, Guanjun; Li, Yan; Yuan, Guiyong; Chen, Yong; Lei, Ming; Huang, Jing
      Nature (London, United Kingdom) (2019), 573 (7774), 445-449CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Methyltransferases of the mixed-lineage leukemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have crit. and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are assocd. with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 Me marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favors open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules assocd. with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the assocn. between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.
    35. 35
      Dharmarajan, V.; Lee, J. H.; Patel, A.; Skalnik, D. G.; Cosgrove, M. S. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases. J. Biol. Chem. 2012, 287, 27275– 27289,  DOI: 10.1074/jbc.M112.364125
      35
      Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases
      Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G.; Cosgrove, Michael S.
      Journal of Biological Chemistry (2012), 287 (33), 27275-27289CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are assocd. with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoppt. WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we detd. structures of WDR5 bound to six different naturally occurring Win motif sequences at resolns. ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.
    36. 36
      Zhang, P.; Lee, H.; Brunzelle, J. S.; Couture, J. F. The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases. Nucleic Acids Res. 2012, 40, 4237– 4246,  DOI: 10.1093/nar/gkr1235
      36
      The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases
      Zhang, Pamela; Lee, Hwabin; Brunzelle, Joseph S.; Couture, Jean-Francois
      Nucleic Acids Research (2012), 40 (9), 4237-4246CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochem. and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative anal. of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.
    37. 37
      Weeramange, C. J.; Fairlamb, M. S.; Singh, D.; Fenton, A. W.; Swint-Kruse, L. The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules. Protein Sci. 2020, 29, 1004,  DOI: 10.1002/pro.3827
      There is no corresponding record for this reference.
    38. 38
      Masson, J. F. Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics. ACS Sens 2017, 2, 16– 30,  DOI: 10.1021/acssensors.6b00763
      38
      Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics
      Masson, Jean-Francois
      ACS Sensors (2017), 2 (1), 16-30CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      A review. The design and application of sensors for monitoring biomols. in clin. samples is a common goal of the sensing research community. Surface plasmon resonance (SPR) and other plasmonic techniques such as localized surface plasmon resonance (LSPR) and imaging SPR are reaching a maturity level sufficient for their application in monitoring biomols. in clin. samples. In recent years, the first examples for monitoring antibodies, proteins, enzymes, drugs, small mols., peptides, and nucleic acids in biofluids collected from patients afflicted with a series of medical conditions (Alzheimer's, hepatitis, diabetes, leukemia, and cancers such as prostate and breast cancers, among others) demonstrate the progress of SPR sensing in clin. chem. This Perspective reviews the current status of the field, showcasing a series of early successes in the application of SPR for clin. anal. and detailing a series of considerations regarding sensing schemes, exposing issues with anal. in biofluids, and comparing SPR with ELISA, while providing an outlook of the challenges currently assocd. with plasmonic materials, instrumentation, microfluidics, bioreceptor selection, selection of a clin. market, and validation of a clin. assay for applying SPR sensors to clin. samples. Research opportunities are proposed to further advance the field and transition SPR biosensors from research proof-of-concept stage to actual clin. applications.
    39. 39
      Pang, X.; Zhou, H. X. Rate Constants and Mechanisms of Protein-Ligand Binding. Annu. Rev. Biophys. 2017, 46, 105– 130,  DOI: 10.1146/annurev-biophys-070816-033639
      39
      Rate Constants and Mechanisms of Protein-Ligand Binding
      Pang, Xiaodong; Zhou, Huan-Xiang
      Annual Review of Biophysics (2017), 46 (), 105-130CODEN: ARBNCV; ISSN:1936-122X. (Annual Reviews)
      Whereas protein-ligand binding affinities have long-established prominence, binding rate consts. and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new exptl. techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate consts., can and should be quant. detd. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and phys. insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its redn. to simple rate equations; computation of binding rate consts.; quant. detn. of binding mechanisms; and elucidation of phys. factors that control binding rate consts. and mechanisms.
    40. 40
      Nitzan, A. Chemical Dynamics in Condensed Phases; Oxford University Press: 2006.
      There is no corresponding record for this reference.
    41. 41
      Zhou, H. X. Rate theories for biologists. Q. Rev. Biophys. 2010, 43, 219– 293,  DOI: 10.1017/S0033583510000120
      41
      Rate theories for biologists
      Zhou Huan-Xiang
      Quarterly reviews of biophysics (2010), 43 (2), 219-93 ISSN:.
      Some of the rate theories that are most useful for modeling biological processes are reviewed. By delving into some of the details and subtleties in the development of the theories, the review will hopefully help the reader gain a more than superficial perspective. Examples are presented to illustrate how rate theories can be used to generate insight at the microscopic level into biomolecular behaviors. An attempt is made to clear up a number of misconceptions in the literature regarding popular rate theories, including the appearance of Planck's constant in the transition-state theory and the Smoluchowski result as an upper limit for protein-protein and protein-DNA association rate constants. Future work in combining the implementation of rate theories through computer simulations with experimental probes of rate processes, and in modeling effects of intracellular environments so that theories can be used for generating rate constants for systems biology studies is particularly exciting.
    42. 42
      Smoluchowski, M. Mathematical Theory of the Kinetics of the Coagulation of Colloidal Solutions. Z. Phys. Chem. 1917, 92, 129– 135
      There is no corresponding record for this reference.
    43. 43
      Hanggi, P.; Talkner, P.; Borkovec, M. Reaction-Rate Theory - 50 Years After Kramers. Rev. Mod. Phys. 1990, 62, 251– 341,  DOI: 10.1103/RevModPhys.62.251
      There is no corresponding record for this reference.
    44. 44
      Berg, H. C.; Purcell, E. M. Physics of chemoreception. Biophys. J. 1977, 20, 193– 219,  DOI: 10.1016/S0006-3495(77)85544-6
      44
      Physics of chemoreception
      Berg, Howard C.; Purcell, Edward M.
      Biophysical Journal (1977), 20 (2), 193-219CODEN: BIOJAU; ISSN:0006-3495.
      Statistical fluctuations limit the precision with which a microorganism can, in a given time T, det. the concn. of a chemoattractant in the surrounding medium. The best a cell can do is to monitor continually the state of occupation of receptors distributed over its surface. For nearly optimum performance only a small fraction of the surface need be specifically adsorbing. The probability that a mol. that has collided with the cell will find a receptor is Ns/(Ns + πa), if N receptors, each with a binding site of radius s, are evenly distributed over a cell of radius a. There is ample room for many independent systems of specific receptors. The adsorption rate for mols. of moderate size cannot be significantly enhanced by motion of the cell or by stirring of the medium by the cell. The no. of specific receptors needed to attain such precision is about a/s. Data on bacteriophage adsorption, bacterial chemotaxis, and chemotaxis in a cellular slime mold are evaluated. The chemotactic sensitivity of Escherichia coli approaches that of the cell of optimum design.
    45. 45
      Misiura, M. M.; Kolomeisky, A. B. Role of Intrinsically Disordered Regions in Acceleration of Protein-Protein Association. J. Phys. Chem. B 2020, 124, 20– 27,  DOI: 10.1021/acs.jpcb.9b08793
      45
      Role of Intrinsically Disordered Regions in Acceleration of Protein-Protein Association
      Misiura, Mikita M.; Kolomeisky, Anatoly B.
      Journal of Physical Chemistry B (2020), 124 (1), 20-27CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Although Intrinsically Disordered Proteins (IDPs) and Intrinsically Disordered Regions (IDRs) in folded proteins are not able to form stable structures, it is known that they play critically important roles in various biol. processes. However, despite multiple studies, the mol. mechanisms of their functioning remain not fully understood. In this work, we theor. investigate the role of IDRs in acceleration of protein-protein assocn. processes. Our hypothesis is that in protein pairs with several independent binding sites the assocn. process goes faster if some of these binding sites are located on IDRs or connected by IDRs. To test this idea, we employed anal. modeling, numerical calcns. and Brownian Dynamics computer simulations to calc. protein-protein assocn. reaction rates for ERK2-EtsΔ138 system, belonging to RAS-RAF-MEK-ERK signaling pathway in living cells. It is found that putting a binding site on IDR accelerates assocn. process by a factor of 3 to 4. Possible mol. explanations of these observations are presented, and other systems that might use this mechanism are also mentioned.
    46. 46
      Schreiber, G.; Haran, G.; Zhou, H. X. Fundamental aspects of protein-protein association kinetics. Chem. Rev. 2009, 109, 839– 860,  DOI: 10.1021/cr800373w
      46
      Fundamental Aspects of Protein-Protein Association Kinetics
      Schreiber, G.; Haran, G.; Zhou, H.-X.
      Chemical Reviews (Washington, DC, United States) (2009), 109 (3), 839-860CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. This review focuses on recent advances in deciphering the kinetic pathway of protein complex formation, the nature of the precomplex formed through diffusion (which the authors term the "transient complex"), the transition state and other intermediates (such as the so-called encounter complex) along the assocn. pathway.
    47. 47
      Shoemaker, B. A.; Portman, J. J.; Wolynes, P. G. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 8868– 8873,  DOI: 10.1073/pnas.160259697
      47
      Speeding molecular recognition by using the folding funnel: the fly-casting mechanism
      Shoemaker, Benjamin A.; Portman, John J.; Wolynes, Peter G.
      Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (16), 8868-8873CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein mol. can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the "fly-casting mechanism.". We illustrate this scenario with the hypothetical kinetics of binding a single repressor mol. to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.
    48. 48
      Levy, Y.; Onuchic, J. N.; Wolynes, P. G. Fly-casting in protein-DNA binding: frustration between protein folding and electrostatics facilitates target recognition. J. Am. Chem. Soc. 2007, 129, 738– 739,  DOI: 10.1021/ja065531n
      48
      Fly-casting in protein-DNA binding: frustration between protein folding and electrostatics facilitates target recognition
      Levy, Yaakov; Onuchic, Jose N.; Wolynes, Peter G.
      Journal of the American Chemical Society (2007), 129 (4), 738-739CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Mol. plasticity, the key to many biomol. self-assembly processes, and electrostatic steering, which guides proteins to DNA, are shown to be coupled and to facilitate DNA search. While protein flexibility is involved in induced-fit recognition and to a larger extent in intrinsically unstructured DNA binding proteins, we show that through a "tidal force" the electrostatic field of the DNA can induce flexibility and the partial unfolding of a two-state folding protein, thereby reducing its folding barrier and, thus, stimulating fly-casting. The protein binds DNA nonspecifically in a partially folded state and completes its folding when it binds the specific site. The interplay between fly-casting and electrostatics is obsd. even for weak electrostatic forces and is expected to vary with the electrostatic screening due to salt and the intrinsic folding barrier, both of which can be modulated exptl.
    49. 49
      Trizac, E.; Levy, Y.; Wolynes, P. G. Capillarity theory for the fly-casting mechanism. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 2746– 2750,  DOI: 10.1073/pnas.0914727107
      49
      Capillarity theory for the fly-casting mechanism
      Trizac, Emmanuel; Levy, Yaakov; Wolynes, Peter G.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (7), 2746-2750CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Biomol. folding and function are often coupled. During mol. recognition events, one of the binding partners may transiently or partially unfold, allowing more rapid access to a binding site. We describe a simple model for this fly-casting mechanism based on the capillarity approxn. and polymer chain statistics. The model shows that fly casting is most effective when the protein unfolding barrier is small and the part of the chain which extends toward the target is relatively rigid. These features are often seen in known examples of fly casting in protein-DNA binding. Simulations of protein-DNA binding based on well-funneled native-topol. models with electrostatic forces confirm the trends of the anal. theory.
    50. 50
      Sugase, K.; Dyson, H. J.; Wright, P. E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 2007, 447, 1021– 1025,  DOI: 10.1038/nature05858
      50
      Mechanism of coupled folding and binding of an intrinsically disordered protein
      Sugase, Kenji; Dyson, H. Jane; Wright, Peter E.
      Nature (London, United Kingdom) (2007), 447 (7147), 1021-1025CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Protein folding and binding are analogous processes, in which the protein 'searches' for favorable intramol. or intermol. interactions on a funnelled energy landscape. Many eukaryotic proteins are disordered under physiol. conditions, and fold into ordered structures only on binding to their cellular targets. The mechanism by which folding is coupled to binding is poorly understood, but it has been hypothesized on theor. grounds that the binding kinetics may be enhanced by a 'fly-casting' effect, where the disordered protein binds weakly and non-specifically to its target and folds as it approaches the cognate binding site. Here we show, using NMR titrns. and 15N relaxation dispersion, that the phosphorylated kinase inducible activation domain (pKID) of the transcription factor CREB forms an ensemble of transient encounter complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate to the fully bound state without dissocn. from KIX. The carboxy-terminal helix of pKID is only partially folded in the intermediate, and becomes stabilized by intermol. interactions formed in the final bound state. Future applications of our method will provide new understanding of the mol. mechanisms by which intrinsically disordered proteins perform their diverse biol. functions.
    51. 51
      Wright, P. E.; Dyson, H. J. Linking folding and binding. Curr. Opin. Struct. Biol. 2009, 19, 31– 38,  DOI: 10.1016/j.sbi.2008.12.003
      51
      Linking folding and binding
      Wright, Peter E.; Dyson, H. Jane
      Current Opinion in Structural Biology (2009), 19 (1), 31-38CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)
      A review. Many cellular proteins are intrinsically disordered and undergo folding, in whole or in part, upon binding to their physiol. targets. The past few years have seen an exponential increase in papers describing characterization of intrinsically disordered proteins (IDP), both free and bound to targets. Although NMR spectroscopy remains the favored tool, a no. of new biophys. techniques are proving exceptionally useful in defining the limits of the conformational ensembles. Advances have been made in prediction of the recognition elements in IDP, in elucidating the kinetics and mechanism of the coupled folding and binding process, and in understanding the role of post-translational modifications in tuning the biol. response. Here, the authors review these and other recent advances that are providing new insights into the conformational propensities and interactions of IDP and are beginning to reveal general principles underlying their biol. functions.
    52. 52
      Soranno, A.; Koenig, I.; Borgia, M. B.; Hofmann, H.; Zosel, F.; Nettels, D.; Schuler, B. Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 4874– 4879,  DOI: 10.1073/pnas.1322611111
      52
      Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments
      Soranno, Andrea; Koenig, Iwo; Borgia, Madeleine B.; Hofmann, Hagen; Zosel, Franziska; Nettels, Daniel; Schuler, Benjamin
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (13), 4874-4879CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-mol. Foerster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concn., but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the obsd. behavior can be explained quant. if the polymeric nature of both the IDPs and the crowding mols. is taken into account explicitly. Our results suggest that excluded vol. interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.
    53. 53
      Mollica, L.; Bessa, L. M.; Hanoulle, X.; Jensen, M. R.; Blackledge, M.; Schneider, R. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment. Front. Mol. Biosci. 2016, 3, 52,  DOI: 10.3389/fmolb.2016.00052
      53
      Binding mechanisms of intrinsically disordered proteins: theory, simulation, and experiment
      Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjoebing; Blackledge, Martin; Schneider, Robert
      Frontiers in Molecular Biosciences (2016), 3 (), 52/1-52/18CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
      A review. In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an assocd. function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiol. partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the "fly-casting" hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Exptl., these questions remain difficult to address. Here, we review exptl. studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theor. concepts and numerical methods that can be applied to describe these interactions at the at. level. The available literature suggests that the kinetic and thermodn. parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes obsd. exptl. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.
    54. 54
      Umezawa, K.; Ohnuki, J.; Higo, J.; Takano, M. Intrinsic disorder accelerates dissociation rather than association. Proteins 2016, 84, 1124– 1133,  DOI: 10.1002/prot.25057
      54
      Intrinsic disorder accelerates dissociation rather than association
      Umezawa, Koji; Ohnuki, Jun; Higo, Junichi; Takano, Mitsunori
      Proteins: Structure, Function, and Bioinformatics (2016), 84 (8), 1124-1133CODEN: PSFBAF; ISSN:1097-0134. (Wiley-Blackwell)
      The intrinsically disordered protein (IDP) has distinct properties both phys. and biol.: it often becomes folded when binding to the target and is frequently involved in signal transduction. The phys. property seems to be compatible with the biol. property where fast assocn. and dissocn. between IDP and the target are required. While fast assocn. has been well studied, fueled by the fly-casting mechanism, the dissocn. kinetics has received less attention. Here, the authors studied how intrinsic disorder affects the dissocn. kinetics, as well as the assocn. kinetics, paying attention to the interaction strength at the binding site (i.e., the quality of the "fly lure"). Coarse-grained mol. dynamics simulation of the pKID-KIX system (pKID is an intrinsically disordered region of CREB protein and KIX is the KID-interaction domain od CREB-binding protein), a well-studied IDP system, shows that the assocn. rate becomes larger as the disorder-inducing flexibility that was imparted to the model is increased, but the acceleration is marginal and turns into deceleration as the quality of the fly lure is worsened. In contrast, the dissocn. rate is greatly enhanced as the disorder is increased, indicating that intrinsic disorder serves for rapid signal switching more effectively through dissocn. than assocn.
    55. 55
      Huang, Y.; Liu, Z. Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the “fly-casting” mechanism. J. Mol. Biol. 2009, 393, 1143– 1159,  DOI: 10.1016/j.jmb.2009.09.010
      55
      Kinetic Advantage of Intrinsically Disordered Proteins in Coupled Folding-Binding Process: A Critical Assessment of the "Fly-Casting" Mechanism
      Huang, Yongqi; Liu, Zhirong
      Journal of Molecular Biology (2009), 393 (5), 1143-1159CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Intrinsically disordered proteins (IDPs) are recognized to play important roles in many biol. functions such as transcriptional and translational regulation, cellular signal transduction, protein phosphorylation, and mol. assemblies. The coupling of folding with binding through a "fly-casting" mechanism has been proposed to account for the fast binding kinetics of IDPs. In this article, exptl. data from the literature were collated to verify the kinetic advantages of IDPs, while mol. simulations were performed to clarify the origin of the kinetic advantages. The phosphorylated KID-kinase-inducible domain interacting domain (KIX) complex was used as an example in the simulations. By modifying a coarse-grained model with a native-centric Go-like potential, we were able to continuously tune the degree of disorder of the phosphorylated KID domain and thus investigate the intrinsic role of chain flexibility in binding kinetics. The simulations show that the "fly-casting" effect is not only due to the greater capture radii of IDPs. The coupling of folding with binding of IDPs leads to a significant redn. in binding free-energy barrier. Such a redn. accelerates the binding process. Although the greater capture radius has been regarded as the main factor in promoting the binding rate of IDPs, we found that this parameter will also lead to the slower translational diffusion of IDPs when compared with ordered proteins. As a result, the capture rate of IDPs was found to be slower than that of ordered proteins. The main origin of the faster binding for IDPs are the fewer encounter times required before the formation of the final binding complex. The roles of the interchain native contacts fraction (Qb) and the mass-center distance (ΔR) as reaction coordinates are also discussed.
    56. 56
      Segall, D. E.; Nelson, P. C.; Phillips, R. Volume-exclusion effects in tethered-particle experiments: bead size matters. Phys. Rev. Lett. 2006, 96, 088306,  DOI: 10.1103/PhysRevLett.96.088306
      56
      Volume-Exclusion Effects in Tethered-Particle Experiments: Bead Size Matters
      Segall, Darren E.; Nelson, Philip C.; Phillips, Rob
      Physical Review Letters (2006), 96 (8), 088306/1-088306/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      We give a theor. anal. of bead motion in tethered-particle expts., a single-mol. technique that has been used to explore the dynamics of a variety of macromols. of biol. interest. Our anal. reveals that the proximity of the tethered bead to a nearby surface gives rise to a vol.-exclusion effect, resulting in an entropic stretching-force on the mol. that changes its statistical properties. In addn., vol. exclusion brings about intriguing scaling relations between key observables (statistical moments of the bead) and parameters such as bead size and contour length of the mol. We present analytic and numerical results for these effects in both flexible and semiflexible tethers. Finally, our results give a precise, exptl. testable prediction for the probability distribution of the bead center measured from the polymer attachment point.
    57. 57
      Makarov, D. E. Perspective: Mechanochemistry of biological and synthetic molecules. J. Chem. Phys. 2016, 144, 030901,  DOI: 10.1063/1.4939791
      57
      Perspective: Mechanochemistry of biological and synthetic molecules
      Makarov, Dmitrii E.
      Journal of Chemical Physics (2016), 144 (3), 030901/1-030901/13CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      Coupling of mech. forces and chem. transformations is central to the biophysics of mol. machines, polymer chem., fracture mechanics, tribol., and other disciplines. As a consequence, the same phys. principles and theor. models should be applicable in all of those fields; in fact, similar models have been invoked (and often repeatedly reinvented) to describe, for example, cell adhesion, dry and wet friction, propagation of cracks, and action of mol. motors. This perspective offers a unified view of these phenomena, described in terms of chem. kinetics with rates of elementary steps that are force dependent. The central question is then to describe how the rate of a chem. transformation (and its other measurable properties such as the transition path) depends on the applied force. I will describe phys. models used to answer this question and compare them with exptl. measurements, which employ single-mol. force spectroscopy and which become increasingly common. Multidimensionality of the underlying mol. energy landscapes and the ensuing frequent misalignment between chem. and mech. coordinates result in a no. of distinct scenarios, each showing a nontrivial force dependence of the reaction rate. I will discuss these scenarios, their commonness (or its lack), and the prospects for their exptl. validation. Finally, I will discuss open issues in the field. (c) 2016 American Institute of Physics.
    58. 58
      Imran, A.; Moyer, B. S.; Canning, A. J.; Kalina, D.; Duncan, T. M.; Moody, K. J.; Wolfe, A. J.; Cosgrove, M. S.; Movileanu, L. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions. Biochem. J. 2021, 478, 2145– 2161,  DOI: 10.1042/BCJ20210253
      58
      Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions
      Imran, Ali; Moyer, Brandon S.; Canning, Ashley J.; Kalina, Dan; Duncan, Thomas M.; Moody, Kelsey J.; Wolfe, Aaron J.; Cosgrove, Michael S.; Movileanu, Liviu
      Biochemical Journal (2021), 478 (11), 2145-2161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Recent advances in quant. proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their phys. assocns. with other proteins are ambiguous. A quant. understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient assocns. between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow assocn. kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissocn. kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnol.

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    • Peptide synthesis, purification, and analysis, protein expression and purification, biolayer interferometry, surface plasmon resonance, examples of BLI sensorgrams, determinations of the kinetic and equilibrium constants, scatter plots of the association rate constants versus the dissociation rate constants, examples of SPR sensorgrams, 3D plots and contour maps of the association and dissociation rate constants, supporting references (PDF)


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