Modulating Phase Behavior in Fatty Acid-Modified Elastin-like Polypeptides (FAMEs): Insights into the Impact of Lipid Length on Thermodynamics and Kinetics of Phase Separation

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid–liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous two-phase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7–C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein–lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

* The large standard error for the fitted parameters indicates that this empirical model is not a good descriptor of changes in Tph as a function of lipid length for FAMEs at concentrations below the critical micellization concentration (Figure S10).
Table S3.Apparent activation was derived from the analysis of the ATPS rates using the Arrhenius model.(c,d) This trend persists with further increases in lipid length.Notably, for l≥ 14, the monomer peak is replaced by a slightly larger peak, corresponding to the oligomeric species.

Figure S1 .
Figure S1.The chromatogram obtained for the first five cycles of Edman sequencing of recombinantly expressed ELP (a-e).Edman sequencing confirmed that the N-terminal sequence was GVGVP, consistent with the removal of the initial methionine residue.

Figure S2 .
Figure S2.MALDI-TOF-MS spectra of the proteins used in this study.Vertical dotted lines denote each construct's theoretical molecular weight ([M + H] + ).

Figure S3 .
Figure S3.Representative turbidimetry plots used to determine phase-separation temperatures (Tph) in Figure 3. (a) The turbidity of C12 FAME at different concentrations is plotted as a function of temperature.The absorbance sharply increased as the temperature was increased above Tph.(b) The Tph at each concentration was determined by identifying the peak in the first derivative plots.An arrow denotes the representative Tph for C12 FAME at 100 μM.

Figure S4 .
Figure S4.Reversible phase transition of representative FAMEs.The variable-temperature turbidimetry was conducted at (a) 25 μM; and (b) 100 μM.The phase-separation was found to be completely reversible, regardless of the lipid length.

Figure S5 .
Figure S5.Representative workflow for processing thermal gradient microfluidics (TGM) data of C9 at 310.6 K. (a) The raw Intensity was obtained from the light-scattering data as a function of time at each temperature region.The data for the first 60 s (before the red dashed line), corresponding to the equilibration time of the temperature gradient, were discarded.(b)Data were baseline-corrected by subtracting the linear baseline corresponding to the average intensity of the pixels at 600-1000 s, corresponding to the time required for completion of the ATPS.(c) The data was normalized using the maximum intensity and background intensity.We plotted the light-scattering intensity for each temperature range within a 1-10 minute timeframe.Data after 10 minutes were discarded as there was little change in intensity after that point.After normalizing the data, it was fitted into either a first-order or second-order model.The figure illustrates a representative fit to the first-order model using a single-exponential decay, as shown by the red solid line.The dashed line represents the 95% confidence interval.

Figure S6 . 2 Figure
Figure S6.The retention time of FAMEs vs. the number of methylene groups can be well-described as a quadratic equation.The x-axis represents the number of methylene groups in the lipid, ranging from zero in C2 to 14 in C16.Such a relationship has been previously observed in the literature for modeling the elution time of fatty acids and hydrophobic peptides.1, 2

Figure S8 .
Figure S8.Concentraiton-dependence of the Curve fitting parameters correlating Tph to lipid length.(a) The plot displays the Tt and Tc parameters, which are obtained from the 4PL (four-parameter logistic) model and indicated by green and red symbols, respectively.These parameters are compared with the predicted transition temperature of an unmodified ELP with a similar guest residue at two limits: 1) at similar concentration and length, as indicated by the blue dashed line; 2) at the high molecular weight limit, based on the model established by McDaniel et al. 3 (b) The hill slope(s) was approximately -0.2, although it showed a slightly negative correlation with the concentration.(c) The sigmoid's midpoint (lm) was ~ 9-10 for concentrations higher than CMC (Figure S10).

Figure S10 . 4 Figure S11 .
Figure S10.The variation of the critical micelle concentration (CMC) as a function of lipid length.The error bar represents the 95% confidence intervals of the intersections of the lines plotted in FigureS9.Specifically, neither the unmodified ELP nor C2 exhibited any measurable CMC up to 200 µM.However, for the remaining constructs, the CMC decreased from 40 to 3 µM as the lipid length increased from C3 to C16, consistent with prior reports.4

Figure S13 .
Figure S13.Kinetic analysis of ATPS formation in ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S14 .
Figure S14.Kinetic analysis of ATPS formation in C2-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S15 .
Figure S15.Kinetic analysis of ATPS formation in C3-ELP.(a) Dark-field images of the microfluidic channel placed on a linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S16 .
Figure S16.Kinetic analysis of ATPS formation in C4-ELP.(a) Dark-field images of the microfluidic channel placed on a linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S17 .
Figure S17.Kinetic analysis of ATPS formation in C5-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S18 .
Figure S18.Kinetic analysis of ATPS formation in C6-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S19 .
Figure S19.Kinetic analysis of ATPS formation in C7-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S20 .
Figure S20.Kinetic analysis of ATPS formation in C8-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S21 .
Figure S21.Kinetic analysis of ATPS formation in C9-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of three measurements.Concentration is 10 mg/mL.

Figure S22 .
Figure S22.Kinetic analysis of ATPS formation in C10-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of intensity decay to a single-exponential decay function at three temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Figure S23 .
Figure S23.Kinetic analysis of ATPS formation in C11-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the four temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Figure S24 .
Figure S24.Kinetic analysis of ATPS formation in C12-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the four temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Figure S25 .
Figure S25.Kinetic analysis of ATPS formation in C13-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the four temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Figure S26 .
Figure S26.Kinetic analysis of ATPS formation in C14-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the four temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Figure S27 .
Figure S27.Kinetic analysis of ATPS formation in C15-ELP.(a) Dark-field images of microfluidic channel placed on linear temperature gradient at t = 1, 3, and 10 min.(b) 3D plot of the scattering intensity as a function of time and temperature.(c) Representative curve fits of the intensity decay to a single-exponential decay function at the four temperatures.(d) Rate constant as a function of temperature.Error bars represent the standard deviations of six measurements.Concentration is 10 mg/mL.

Table S1 .
Theoretical molecular weight and observed m/z of each construct.

Table S2 .
Curve fitting parameters and goodness-of-fit statistics for a four-parameter logistic curve (4PL) correlating Tph to lipid length calculated for FAMEs at various concentrations.These values are also plotted in FigureS8to faciliate comparison.