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Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors

Cite this: ACS Nano 2021, 15, 7, 12161–12170
Publication Date (Web):June 29, 2021
https://doi.org/10.1021/acsnano.1c03677

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody’s Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.

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Super-resolution microscopy (SRM) enables the investigation of cellular structures with molecular resolution in the nanometer range. (1) An ideal labeling strategy for SRM would achieve site-specific labeling with reporters as close as possible to the target such that the linkage error between epitope and reporter does not worsen the localization accuracy, while allowing for quantitative imaging approaches by a defined stoichiometry. (2) These conditions are best met by genetic fusions of the target protein with a tag, like photoswitchable fluorescent proteins for PALM, or enzymes (SNAP, Halo) that mediate the conjugation with organic fluorophores or DNA handles for DNA-PAINT, respectively. While ectopic expression or CRISP-R Cas mediated genetic modification of many cell lines is straightforward nowadays, (3) altered expression levels as well as the presence of the tag itself can lead to artifacts in the intracellular localization or to changed interactions between the target protein and its cellular environment. (4) Moreover, some cells cannot be genetically modified in vitro, e.g., blood platelets, and tissue or liquid biopsies from patients for biomedical applications require alternative labeling approaches, namely, the usage of affinity tags.
Indirect immunofluorescence is the most common method to label target proteins in cell and tissue samples from patients. Highly target-specific primary antibodies are detected with secondary antibodies modified by reporter fluorophores (for STORM or STED) or DNA strands (for DNA-PAINT). (5) Since nanometer localization precision can be achieved in single-molecule localization microscopy (SMLM), the secondary antibody contributes an additional linkage error between reporter and epitope, which has been estimated to be 10–15 nm, (5−7) ultimately limiting measurement accuracy. Conceptually, linkage errors of antibodies are not well understood; while the antibodies usually bind their targets in a defined orientation resulting in a systematic offset between label and target (inaccuracy), the intramolecular flexibility and the random attachment sites of labels and/or secondary antibodies result in a spread of label positions (imprecision). These two distinct effects are often not distinguished in the literature, and the latter effect is difficult to disentangle from the localization imprecision (as given by the Crámer Rao lower bound, CRLB) and from uncompensated drift. Moreover, the quantification of the number of target proteins is complicated by the random number of modifications on the antibody. (5,20) Although more and more fluorescently labeled primary antibodies are becoming commercially available, the catalogue is far from complete and lacks primary antibodies labeled with DNA handles for DNA-PAINT. Also, current commercial secondaries for DNA-PAINT are restricted to target species mouse and rabbit and do not offer customizable DNA sequences. Although several promising affinity reagents such as nanobodies, aptamers, or affimers have been developed in order to address these challenges, (6,8−10) they rely on elaborate production techniques that are not routine in every laboratory environment, and their availability is still limited to a small number of targets. We propose that site-specific and stoichiometric labeling of immunoglobulins G (IgGs) with a simple protocol, which can be carried out in any lab, could offer a useful alternative for defined target labeling for SRM.
Antibody functionalization at specific sites and with a defined number of reagents is widely used for the synthesis of therapeutic antibody–drug conjugates (ADCs). (11) These well-defined ADCs offer targeted delivery of drugs at better defined dosages and therefore create widened therapeutic windows in cancer chemotherapies. (12) Established protocols include glycan trimming, (13,14) localized lysine modification in the vicinity of histidine clusters, (15) or labeling of specific glutamine residues that become accessible upon deglycosylation. (16−18) These strategies hold promise to yield a homogeneous labeling of IgGs, have a high repeatability and minimal interference with the complementarity-determining region (CDR), and allow for the direct labeling of primary antibodies. These features could benefit SRM by achieving labeling closer to the epitope location, aiding colocalization analyses, denser sampling of cellular structures, and improved counting of proteins in supramolecular complexes. However, none of these approaches have been utilized for SRM to date, and their viability to modify nonhuman IgGs remains largely unexplored.

Results/Discussion

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Site-Specific Antibody–Reporter Conjugation Strategy

In this work, we site-specifically label secondary and primary antibodies after deglycosylation using glutamine modification with azides (Scheme 1) following the pioneering work by Schibli and colleagues on ADCs. (17,18) Such modified antibodies serve as a universal platform to couple different reporters via strain-promoted azide–alkyne cycloaddition (SPAAC). (19) The functionalization protocol is based entirely on commercially available reagents. IgG antibodies are deglycosylated with the help of PNGaseF. Once the N-linked glycans are removed, microbial transglutaminase (mTG) catalyzes the cross-linking of amine-polyethylene glycol-azide (H2N-PEG3-N3) to the amide of the now-accessible glutamine at position −2 relative to the deglycosylated IgG site. In a final step, dibenzocyclooctyne (DBCO)-single-stranded DNA (ssDNA) or DIBO-Alexa Fluor (AF)647 is conjugated using SPAAC (Scheme 1, Supplementary Figure S1). All steps occur at neutral pH and under physiological salt concentrations. We combined the first two steps in a one-pot reaction to simplify the protocol. The intermediate and final products are purified by mild centrifugal filtration.

Scheme 1

Scheme 1. Site-specific labeling of IgG antibodies. In a first reaction, N-linked glycans are removed by PNGaseF, and only the available glutamines (Q) at position −2 relative to the deglycosylated sites are modified with H2N-PEG3-N3 catalyzed by microbial transglutaminase (mTG). In a second reaction, fluorophores(-DIBO) or ssDNA(-DBCO) are attached to the azide-modified antibodies using strain-promoted azide–alkyne cycloaddition (SPAAC).
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The conjugates were evaluated regarding the modification of a single glutamine per heavy chain with the help of mass spectrometry (Supplementary Figure S2) and regarding the degree of labeling (DOL) using UV–vis spectroscopy, which yields the average number of reporters per antibody. The conjugation of secondary antibodies raised in donkey resulted in two modifications per antibody for both DNA labels and fluorescent dye labels (Supplementary Table S1). The DOL moreover did not increase further with a higher excess of H2N-PEG3-N3 (80–300×) or of the reactive SPAAC probe (10–20×) (Supplementary Table S2). This result confirms previous reports using human IgG1 that found that mTG is specific to only one glutamine in each IgG heavy chain (17) and that a sufficient excess of reagents can saturate these sites.

Modification of Different IgG Subtypes from Different Species

To explore the suitability of this method to modify IgGs from different host species in defined ways, we performed a sequence alignment (Figure 1) and tested our protocol for selected IgGs (Supplementary Table S1). The main N-linked glycosylation site is strictly preserved across species and IgG subtypes. The glutamine at position −2 with respect to this glycosylation site is preserved for all human IgG subclasses (IgG1, IgG2, IgG3, IgG4), mouse subclasses IgG1 and IgG3, all rat IgG subclasses (IgG1, IgG2a, IgG2b), in guinea pig (IgG2), and in equine species (IgG1 of horse or donkey). Accordingly, we found good compatibility with all tested donkey secondaries, two mouse IgG1 primaries, two rat IgG2a primaries, a mix of guinea pig IgG, and two humanized IgG1 primaries. The sequence analysis is also in agreement with previous successful modifications of human IgG2, IgG3, and IgG4 (P.R.S., personal communication). Mouse IgG3 has a second glycosylation site (N322) with an additional glutamine at position −2, which theoretically yields 4 instead of 2 possible modification sites per molecule (not tested). Rabbit IgG contains an additional glutamine at position −3 next to the conserved glutamine, and a sequence conflict was reported for the glycosylation site itself. Conjugation attempts with a rabbit primary or with rabbit secondaries showed negligible conjugation efficiency, which suggests that the glutamines are not accessible and/or modified. Manatee IgG and mouse IgG2 subtypes lack the buried glutamine and thus are not susceptible for labeling by this strategy, which was verified using one mouse IgG2b primary (Supplementary Table S1). No sequence information was found for goat or sheep IgGs; our conjugation attempts with goat secondaries resulted in ∼1 modification per IgG, which suggests either incomplete deglycosylation and/or a mixed population of different IgG subtypes. For compatible IgGs, no systematic differences were observed for DIBO–AF647 vs DBCO–DNA coupling efficiency. In summary, the conjugation method strictly depends on the conservation of the glutamine near the N-linked glycosylation site rather than on an IgG isotype or host species in general, and it is compatible with a wide range of antibodies.

Figure 1

Figure 1. Sequence alignment and evaluation of potential modification sites for different IgG subclasses from different host species. Sequences were sourced from the UniProt database and aligned with respect to the N-linked glycosylation sites (gray background). The glutamine at position −2 (black background) is preserved across the majority of IgG subtypes and species for which sequence information was available. *Potential conflict with additional glutamine marked in red; Fl = Florida.

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Microtubule Labeling Shell Dimensions in STORM

We benchmarked the fluorescent AF647-labeled antibodies for 3D STORM in fixed U2OS cells (Figure 2a–d) using microtubules as an in situ standard with known dimensions. We compared three cases: indirect immunolabeling with randomly labeled secondary antibodies (Figure 2b; NHS), indirect immunolabeling with our site-specifically-labeled secondary antibodies (Figure 2c; 2°), or direct immunolabeling with our site-specifically-labeled primaries (Figure 2d; 1°). The number of localizations per micron along microtubules decreased from the randomly labeled secondary (2159 ± 388 μm–1, mean ± std) over the site-specifically-labeled secondary (1634 ± 359 μm–1) to the primary (547 ± 136 μm–1), as expected from the effective number of fluorophores per bound primary. Directly labeled microtubules appeared distinctly thinner than indirectly labeled ones, and single antibodies were clearly discernible by the crisp appearance of localization clusters.

Figure 2

Figure 2. 3D SMLM imaging of microtubule networks and determination of labeling shell dimensions for indirect and direct immunolabeling. (a–d) 3D STORM using Alexa Fluor 647 labeled antibodies in fixed U2OS cells. (a) Schematic (not to scale). (b) Indirect immunolabeling using a randomly labeled secondary donkey-anti-mouse (NHS 2°). (c) Indirect immunolabeling using a site-specifically-labeled secondary donkey-anti-mouse (2°). (d) Direct immunostaining using a site-specifically-labeled primary mouse-anti-α-tubulin (1°). (e–h) 3D DNA-PAINT using DNA-labeled antibodies in U2OS cells (f) and human platelets (g, h). (e) Schematic (not to scale). (f, g) Indirect immunolabeling using a site-specifically-labeled donkey-anti-mouse secondary (2°). (h) Direct labeling using a site-specifically-labeled primary mouse-anti-α-tubulin (1°). For all conditions, representative 3D SMLM images are shown (left). Labeling shell dimensions around microtubules were determined from averaged experimental yz cross-sections and fitted label distributions (right). The fitted label distribution was a Gaussian ring kernel (r: radius; w: full width at half-maximum) convolved with the localization precisions in y and z, respectively. (i) Comparison of center positions and widths of labeling shell dimensions for the different labeling strategies in b–d and f–h.

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To quantify the linkage error that antibodies add to the microtubule dimension, we fitted the apparent dimensions of the antibody labeling shell around microtubules taking into account the thickness of the shell and in addition the blur by the localization precision (see Methods/Experimental). The averaged cross-sectional profiles and results are shown on the right of Figure 2b–d, and the derived dimensions are summarized in Table 1. The average outer radius of microtubules is 12.5 nm. The radius/center of the fluorophore-containing shell around microtubules was 22.5 ± 0.2 nm (best fit ±95% confidence interval) for randomly labeled secondaries, 22.5 ± 0.2 nm for site-specifically-labeled secondaries, and 20.2 ± 0.2 nm for site-specifically-labeled primaries. The thickness of the shell resulted as 18.1 ± 0.3, 16.9 ± 0.3, and 10.0 ± 0.5 nm, respectively. Dimensions were significantly different between the site-specifically-labeled 2° antibody and the 1° antibody (two-sided F-test; radius: F = 30.8, p < 0.0001; thickness: F = 27.7, p < 0.0001). Commercial secondary antibodies yielded the same radius (F = 0.2, p = 0.66) but a slightly thicker shell (F = 9.2, p = 0.0029) compared to the site-specifically-labeled secondaries, in agreement with previous findings, which found that immunostained microtubules were less well resolved when using antibodies with a higher degree of labeling. (20) While these derived shell dimensions might be affected by nonstraight microtubules, imperfect drift correction, and/or imperfect registration, these errors should be comparable for different experiments. The comparison thus shows that direct immunofluorescence significantly decreases the offset to the epitope (thus, inaccuracy) by 2.3 nm and the spread of label positions (thus, precision) by ∼40% as compared to indirect immunofluorescence. Please note that the DOL of 1° and 2° antibodies was comparable due to our consistent labeling technique, which rules out the DOL as a potential confounding factor. (20) The maximum linkage error (Table 1) thus was ∼19 nm for indirect and ∼13 nm for direct labeling. Importantly, the linkage errors of primary and secondary antibodies were not strictly additive. The obtained diameters of labeling shells (∼45 nm for secondary antibodies, ∼40 nm for primary antibodies) may be compared to previously reported apparent microtubules dimensions from 2D STORM images measured by the full-width-at-half-maximum (fwhm) of a single Gaussian fit to intensity line profiles perpendicular to the microtubule for secondary antibodies (59.5 nm, (20) 61.7 nm (8)), primary antibodies (54.0 nm (8)), or secondary nanobodies (37.5 nm, (21) 39.3 nm (8)) or by measuring the peak-to-peak distance of a double Gaussian fit using secondary antibodies (35 nm (22)) or secondary nanobodies (∼32 nm, (23) 30–40 nm (24)). Please note that these derived values still contain systematic errors due to the z-projection, localization imprecision, and oversimplified fitting models.
Table 1. Measured Dimensions of the Microtubule Labeling Shell of Different Secondary (NHS, 2°) or Primary (1°) Antibody–Reporter Conjugates Using STORM (“647”) or DNA-PAINT (“DNA”), Respectivelya
cellsantibody labelingshell radius r (nm)bthickness w (nm)bminimum linkage errorc (nm)maximum linkage errord (nm)
U2OSαTub + αMs-647 NHS 2°22.5 ± 0.1818.1 ± 0.271.0 ± 0.3219.1 ± 0.32
U2OSαTub + αMs-647 2°22.5 ± 0.2216.9 ± 0.341.6 ± 0.4118.5 ± 0.41
U2OSαTub-647 1°20.2 ± 0.2410.0 ± 0.482.7 ± 0.5412.7 ± 0.54
U2OSαTub + αMs-DNA 2°31.8 ± 0.1421.6 ± 0.198.5 ± 0.2430.1 ± 0.24
human plateletsαTub + αMs-DNA 2°29.2 ± 0.1621.4 ± 0.216.0 ± 0.2627.4 ± 0.26
human plateletsαTub-DNA 1°23.5 ± 0.2016.7 ± 0.282.7 ± 0.3419.4 ± 0.34
a

Parameters were obtained from fits shown in Figure 2.

b

Best fit parameters ±95% confidence intervals.

c

The minimum linkage error was calculated using the formula (r – 12.5 nm) – 0.5w.

d

The maximum linkage error was calculated using the formula (r – 12.5 nm) + 0.5w.

Microtubule Labeling Shell Dimensions in DNA-PAINT

Next, we used the ssDNA-labeled antibodies for DNA-PAINT imaging of microtubules in situ in U2OS cells or in spread human platelets (Figure 2e–h). Similar to STORM, directly labeled microtubules (1°) appeared distinctly thinner compared to indirect immunolabeling (2°), both in the images themselves and in terms of the averaged cross-sections. The radius of the microtubule labeling shell resulted as 31.8 ± 0.14 nm for indirect DNA-PAINT in U2OS cells (reanalysis of previously published data (25)) and 29.2 ± 0.16 nm in platelets (F = 80.9, p < 0.0001) and similar thicknesses of 21.6 ± 0.19 and 21.4 ± 0.21 nm, respectively (F = 3.5, p = 0.0628) (Table 1). Dimensions obtained by direct DNA-PAINT were 23.5 ± 0.20 nm for the radius and 16.7 ± 0.28 nm for the thickness and thus highly significantly smaller than those obtained by indirect DNA-PAINT (radius: F = 129.0, p < 0.0001; thickness: F = 57.7, p < 0.0001). The microtubule diameters of indirect DNA-PAINT (∼59–63 nm) may be compared with previous 2D DNA-PAINT measurements of the peak-to-peak distance of a double Gaussian fit to the perpendicular intensity line profile (∼47 nm (26)). Please note that in a z-projection the peak intensities move inward and a fit tends to underestimate the true labeling shell diameter. The maximum linkage error in our DNA-PAINT measurements thus was ∼27–30 nm for indirect and ∼19 nm for direct labeling, again emphasizing that the linkage errors of primaries and secondaries are not strictly additive.
Unexpectedly, the label shell dimensions in DNA-PAINT were significantly larger than those in STORM, in terms of both radius and thickness of the labeling shells, both for indirect labeling (radius: F = 137.83, p < 0.0001; thickness: F = 44.4, p < 0.0001) and for direct labeling (radius: F = 47.7, p < 0.0001; thickness: F = 30.2, p < 0.0001) (Figure 2i). This was consistently observed in different cell types and on different microscopes, for fitting of pooled cross-sections (Figure 2) as well as of individual microtubule segments (Supplementary Figure S3). Of note, AF647 fluorophores on secondary antibodies reached close to the microtubule surface, whereas DNA docking strands on secondary antibodies were depleted from around the microtubule (see Figure 2i and minimum linkage errors in Table 1). While an underestimation of the localization precision, which was smaller in DNA-PAINT as compared to STORM, might lead to an overestimation of the fitted shell thickness, it has little impact on the fitted radius (Supplementary Figure S4). The same strict filtering criteria regarding deviations from the experimental point spread function were applied, which rules out systematic experimental localization errors. The design of the DNA-PAINT imager strand positioned the fluorophore at the antibody-proximal end of the docking strand, only a C5 linker length (between DBCO and ssDNA, ca. 0.5 nm) away from the epitope, similar to DIBO-AF647. We thus conclude that technical aspects of the two SMLM imaging approaches cannot fully explain the observed difference in the label distribution around microtubules in STORM versus DNA-PAINT experiments.

Linkage Errors in Monte Carlo Simulations of Immunolabeled Microtubules

To better understand the spatial distribution of reporters around immunolabeled microtubules, we performed Monte Carlo simulations of immunolabeled microtubules using a geometric model that takes advantage of the known attachment site of the reporter at IgGs due to our site-specific labeling strategy (Figure 3, Supplementary Tables S3 and S4). The primary antibody binds an epitope on α-tubulin at the outer surface of a microtubule, which was modeled based on cryo-EM data (27) (Figure 3a). Fab and Fc antibody segments were simulated as rigid bodies joined by a flexible hinge, which is a validated and widely accepted assumption. (28,29) Segment lengths were determined from a mouse IgG2a crystal structure, (30) while hinge flexibility was modeled according to the measured conformational distribution of IgG molecules in solution (31) (Figure 3b). The binding sites of polyclonal secondary antibodies were assumed to be evenly distributed over the Fc region of the primary antibody. The reporter was attached to Glu295 via a flexible C5 linker. Steric clashes were excluded by restricting angles between neighboring segments, obeying a minimum distance between non-neighboring segments, and treating the microtubule as impenetrable (see Methods).

Figure 3

Figure 3. Monte Carlo simulations of antibody conformations at microtubules. (a) Location of the epitope of the anti-α-tubulin antibody in our geometric model (right) based on the cryo-EM structure of a microtubule (left; PDB 5SYF, ref (27); molecular surface rendered using Mol*, ref (33)). (b) Each IgG molecule was modeled by two segments corresponding to Fab and Fc fragments connected by a flexible hinge region. Right: Parametrization of Fab, Fc, and linker segments of the labeled primary antibody. Left: Dimensions were based on the crystallographic structure of IgG2a (PDB 1IGT, ref (30), visualization by Mol*). While the location of the modification is precisely known (Glu), the binding sites of polyclonal secondary antibodies are assumed to be evenly distributed over the Fc region. For a schematic of indirect immunolabeling, see Supplementary Figure S5c). Simulated reporter distribution for primary antibodies. Left: yz cross-section. Right: Radial distribution. (d) Simulated reporter distribution for primary plus secondary antibody complexes. Panels as in (c). (e) Comparison of reporter distributions for primary antibodies between simulations (left; convolved with the localization and experimental imprecision) and experiments (middle: pooled cross-sections as in Figure 2). Right: Normalized residuals of the difference between experiment and model. Top: STORM. Bottom: DNA-PAINT. (f) Comparison of reporter distributions for secondary antibodies between simulations and experiments. Panels as in (d).

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The label distribution of simulated primary antibodies peaked at 20.5 nm distance from the microtubule center and had a fwhm of 6.9 nm (Figure 3c). The simulated label distribution for indirect immunolabeling peaked at 22.9 nm and showed a much larger spread of 14.7 nm (Figure 3d). While the peak positions of the label distributions vary with the choice of the unknown epitope location on the microtubule (Supplementary Figure S6) and the unknown rigidity of the Fab-epitope orientation (Supplementary Figure S7) by up to ±1.4 nm, the by ca. 2.4 nm larger peak distance and the by 7.8 nm substantially wider distribution for indirect compared to direct immunolabeling were robust against these parameter changes. This can be rationalized by the following mechanism: The flexible hinge region within the primary antibody largely randomizes the direction into which its Fc segment and accordingly also the secondary antibody protrude. As a consequence, the secondary antibody mainly adds to the spread and much less to the distance of the labels from the target. This explanation for nonadditive errors of primary and secondary antibody layers based on molecular conformational flexibility shares some similarities with a proposed explanation based on mere error propagation, (32) which, however, does not account for the orientation bias of the primary antibody nor distinguish between inaccuracy (systematic offset) and imprecision (spread).

Benchmarking of SMLM Results against Simulations

The relative differences between simulated primary and secondary label distributions in terms of peak position and width favorably compare to the relative differences between the label distributions that resulted from STORM data (Table 1). Moreover, simulated and experimental (STORM) peak positions agreed quantitatively, at least within the uncertainty caused by the choices of unknown parameters (see above). The experimental fwhm was larger by ∼3 nm compared to the simulated width, which could arise from nonstraight microtubules or from an imperfect fit of the axis direction of individual microtubules. When convolved with the experimental localization uncertainties, the simulated cross-sections of primary and secondary label distributions matched the STORM cross-sections very well (Figure 3e,f). We conclude that the results from STORM experiments match the theoretical predictions.
Systematic differences were seen when comparing DNA-PAINT cross-sections to simulations, with a considerable outward shift of the measured labeling shell (Figure 3e,f). Technical reasons could be excluded since STORM imaging of indirectly labeled microtubules using DNA-labeled primary and AF647-labeled secondary antibodies yielded similar results (Supplementary Figure 8a) as DNA-PAINT of indirectly labeled microtubules (Figure 2f,g). We thus proposed that DNA-labeled (primary or secondary) antibodies assumed different conformations than the AF647-labeled antibodies or the simulated antibodies. A possibility is that negative charges of DNA led to a repulsive interaction and a radial alignment of antibodies. Simulations of more straight antibody conformations shifted the peak of the radial label distributions outward and depleted the labels from the microtubule vicinity as in the experiments (cf. Figure 2i); however, they substantially reduced the width of the label distribution and thus systematically underestimated the measured shell thicknesses, for both direct and indirect labeling (Supplementary Figure 8b). Since a mere reorientation could not fully explain the larger shells, we hypothesized that the contour length of the antibody was increased. Deglycosylation is known to render IgGs more prone to chemical or thermal denaturation. (34,35) We speculate that removing positively charged glycans and adding negatively charged DNA in close proximity could potentially destabilize the Fc structure and cause partial unfolding, which would increase the contour length and flexibility. Far-UV circular dichroism (CD) spectra showed negligible changes upon IgG deglycosylation but a more negative ellipticity at 217 nm of DNA–IgG conjugates (Supplementary Figure 8c), comparable to signal changes resulting from other denaturation pathways. (36,37) Since it was not possible to obtain further experimental evidence for IgG partial unfolding upon DNA conjugation, the underlying reason for the larger labeling shells in DNA-PAINT remained incompletely understood.

Conclusions

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Site-specific and quantitative functionalization of antibodies with fluorescent dyes or ssDNA with the help of mTG and subsequent click reactions was highly consistent for the tested IgGs, as opposed to common NHS labeling, which varies more strongly from batch to batch and with the number of reactive lysine residues on different antibodies. Our work thus significantly extends the scope of the original approach (18) by demonstrating its applicability to a range of IgGs from different species and their applications to SRM. Similar functionalization results for a range of other compatible IgG subtypes and primary antibodies can be expected based on sequence analysis except mouse IgG2 and rabbit IgG (Figure 1 and Supplementary Table S1). As a note of caution, the protocol is incompatible with the presence of gelatin, BSA, or primary amines in storage buffers, as these interfere with the enzymatic coupling step, a minor problem that can be overcome by prepurification via protein A/G resin and/or dialysis.
Our precise determination of labeling shell dimensions based on averaged microtubule cross-sections and the modeling of antibody complex conformations allowed an unequivocal interpretation of linkage errors when using antibodies for SRM, summarized in Figures 2i and 3. The excellent agreement between results from STORM and Monte Carlo simulations validates the dimensions of, and the considerable flexibility within, the antibodies. Mapping of the exact epitope location of the used anti α-tubulin antibody could eliminate the remaining uncertainty and further improve theoretical predictions. Secondary antibodies increased the maximum linkage error consistently by 50% less compared to primary antibodies themselves, as a direct consequence of the higher flexibility of the 1°+2° antibody complex than of the 1° alone (Figure 3). Importantly, a purely additive model in which labeling shells behave like impenetrable monolayers is neither consistent with these data nor plausible because the labeling in reality is too sparse to induce interactions between neighboring antibodies. Lastly, the larger outer dimensions in DNA-PAINT compared to STORM require more straightened conformations of 1°+2° antibody complexes and even exceed the theoretical maximum epitope–reporter distance in 1° antibodies. We speculate this could be related to partial loss of IgG quaternary structure upon DNA conjugation.
Our results highlight an unconventional application area for ADC conjugation chemistries for SRM, by providing a straightforward two-step protocol. Our demonstration of labeling and imaging of microtubules in platelets exemplifies the usage of conjugates with cell lines that cannot be genetically engineered and is directly translatable to patient samples. The site-specific modification of glutamines in the Fc region avoids the modification of lysine residues at the CDR and thus provides an alternative approach in cases where amine labeling results in a reduction of binding affinity, a problem variably encountered when labeling primary antibodies. Since the protocol needs neither special reagents nor equipment and comes at ca. 20 € per reaction (Supplementary Table S5), it is easily established in most molecular biology laboratories, as no complicated and costly genetic engineering of antibodies is required. Its adoption could thus benefit many super-resolution microscopists.

Methods/Experimental

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Antibody Labeling

Functionalization reactions were carried out with 50 μg of antibody. The following IgGs were used for SRM: donkey anti-rabbit IgG (Jackson Immuno Research, ref: 711-005-152); donkey anti-mouse IgG (Jackson Immuno Research, ref: 715-005-151); mouse anti-β-tubulin (ThermoFisher Scientific, 32-2500). Other IgGs used for testing the conjugation protocol are listed in Supplementary Table S1. First, 50 μg of antibody was incubated with 0.3 U PNGaseF (Roche, ref: 11365193001), 0.6 U mTG (Zedira, ref: T001), and an 80× molar excess of H2N-PEG3-N3 (click chemistry tools/Jena Bioscience, ref: CLK-AZ101-100) in a one-pot reaction at 37 °C overnight on a shaker at 300 rpm. The unreacted H2N-PEG3-N3 was removed using a centrifugal concentrator (GE Healthcare; Vivaspin 500, 50 kDa nominal molecular weight cutoff) by topping up with PBS to 500 μL and spinning at 7000 rcf for 5 min; this was repeated with freshly added PBS two more times before the sample was recovered. Please note that the dilution with PBS and the relatively large final filtrate volume of ∼40 μL due to reduced centrifugation speed/time ensure that the antibody concentration never significantly exceeded the original concentration (∼1 mg/mL) as a precautionary measure to prevent aggregation. The azide-modified antibodies were then incubated with a 10× molar excess of either DIBO-Alexa Fluor 647 (for STORM; Life Technologies, ref: C10408) or DBCO–DNA (for DNA-PAINT; sequence: DBCO-5′-TTATACATCTA-3′, Biomers) for 2.5 h at RT. The unreacted click reagent was removed using a centrifugal filter as above. Antibodies were stored in PBS pH 7.4 with 1% bovine serum albumin (BSA) and 0.05% sodium azide at 4 °C under exclusion of light for up to 3 months (secondaries) or without BSA at −20 °C for up to 3 years (primaries). Note that pH affects enzyme activity; if the IgG storage buffer is not at physiological pH (∼7.4), pH adjustment prior to the reaction is advisable since a test reaction carried out at pH 6.0 yielded a reduced DOL of ∼0.4–0.5. Should the antibody be prone to aggregation upon deglycosylation, the addition of up to 0.05% Tween 20 to stabilize the antibody in solution is tolerated by the enzymes and does not interfere with reactions.

DOL Characterization

UV–vis absorption on a Nanodrop was used to calculate the average degree of labeling. The DOL for the AF647-conjugated antibodies was determined from the measured absorption values (Nanodrop) at 280 nm (A280) and 650 nm (A650) using the formula DOL = (A650/239 000) × 210 000/(A280 – 0.03 × A650), where 0.03 is the correction factor for AF647 absorption at 280 nm, 239 000 is the molecular extinction coefficient of AF647 at 650 nm, and 210 000 is the molecular extinction coefficient of IgG at 280 nm. The DOL for DNA-conjugated antibodies was determined from the measured absorption values at 280 nm (A280) and 260 nm (A260) using the formulas DOL = cAb/cDNA with cAb = (A280 – A260 × 0.61)/(210 000 – 0.55 × 0.61 × 210‘ 000) and cDNA = (A260 – cAb × 210 000 × 0.55)/142 000, where 0.61 is the correction factor for DNA absorption at 280 nm, 0.55 is the correction factor for protein absorption at 260 nm, 210 000 is the molecular extinction coefficient of IgG at 280 nm, and 142 000 is the molecular extinction coefficient of the P1 handle single-stranded oligo sequence at 260 nm.

Sample Preparation for SMLM

U2OS cells (ATCC HTB-96) were cultured in phenol-red-free DMEM supplemented with 10% fetal calf serum (FCS), NEAA, and Glutamax. Cells were seeded on methanol/HCl-cleaned 24 mm round #1.5 coverslips 2 days before fixation. Washed platelets were purified from whole blood. Blood was obtained from healthy consenting volunteers in line with the declaration of Helsinki and national regulations following ethical approval (RCSI Research Ethics Committee 1394 and 1504). Washed platelets were spread on cleaned 20 mm round #1.5 fibrinogen-coated coverslips in Tyrode’s buffer containing 1.8 mM CaCl2 and 5 μM ADP for 1 h. Both U2OS cells and platelets were briefly washed, extracted in 0.3% glutaraldehyde and 0.25% (v/v) Triton X-100 in cytoskeletal buffer (CB: 10 mM MES pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl2) for 1–2 min, fixed with 2% glutaraldehyde in CB for 10 min, washed, and quenched with 0.1% (w/v) sodium borohydride in PBS for 7 min. Fixed samples were permeabilized (PBS containing 0.1% (v/v) Triton X-100, 0.5% (w/v) bovine serum albumin) for 15 min, washed, blocked (3% BSA in PBS) for 40 min, stained with primary antibodies (10 μg/mL in PBS with 3% BSA) at 4 °C overnight, washed, stained with secondary antibodies (15 μg/mL in PBS with 3% BSA) for 2 h, washed, postfixed (2% paraformaldehyde in PBS), washed, and stored (PBS containing 0.05% sodium azide) at 4 °C in the dark for up to 3 weeks.

Quantification of Apparent Microtubule Dimensions

Individual regions of interest were defined along microtubules, and 200–300 nm long lines were manually drawn in SMAP. (38) The microtubule axis was refined by registering the 3D localizations that lay within <60 nm of the drawn line by least-squares fitting of a hollow 3D cylinder to the data. The registered data were then projected along the cylinder axis. Projected localizations were binned into 2 × 2 nm bins and fitted by a Gaussian ring kernel with ring radius r and sigma σ that was convolved with the anisotropic localization precisions σy (which here stands for the mean of σx and σy) and σz in the lateral and the z-directions, respectively, in the following way. First, to account for the non-normal distribution of CRLB values in either y or z, projected localizations were binned according to their CRLB values in a two-dimensional array, and a compound anisotropic filter kernel was constructed by adding up two-dimensional Gaussians using the binned σy,CRLB and σz,CRLB values as widths and the bin counts as relative weights. Second, to account for the residual uncompensated drift, a two-dimensional Gaussian using the independently determined uncertainties σy,Drift and σz,Drift (see Supplementary Methods) was used as a second filter kernel. The only free fitting parameters were thus r and σ. For the thickness of the labeling shell, we used the fwhm = 2.35 σ of the underlying label distribution. About 150–250 sections along straight parts of microtubules from 2 to 5 cells/separate acquisitions were evaluated per condition.

Monte Carlo Simulations

Monte Carlo simulations were performed and analyzed in MATLAB 2020a (Mathworks). Length measurements of rigid Fab and Fc segments were obtained from the crystal structure of mouse IgG2a (PDB 1IGT) (30) (see Supplementary Table S3). Possible epitope locations on α-tubulin were assessed from the cryo-EM structure of Taxol-stabilized microtubules (PDB 5SYF). (27) Structures were taken from the RSCB database and measured in Mol* (33) (see Supporting Information). Starting from the epitope location (0, y0, z0) and orientation ϕ1 on the microtubule, a kinked chain was constructed characterized by linear segments of lengths Li and rotational angles ϕi and θi around the x-axis and y-axis, respectively, that uniquely defined their orientation with respect to the preceding segment (i > 1) or to the global coordinate system (i = 0; see Figure 3a). Direct immunolabeling was represented by 3 segments (CDR-Hinge, Hinge-Glu, Glu-Reporter; see Figure 3a,b), indirect immunolabeling by 5 segments (1°CDR-1°Hinge, 1°Hinge-2°epitope, 2°CDR-2°Hinge, 2°Hinge-2°Glu, 2°Glu-Reporter; see Supplementary Figure S5). Lengths were either randomly sampled from a finite set of lengths, as measured within the crystal structure, or uniformly picked from a continuous range within defined boundaries (Supplementary Table S4). DBCO–DNA and DIBO–AF647 linkers were modeled as having the same characteristics. A total of 300 000 (primary) or 400 000 (1°+2°) conformations were simulated. Steric hindrance by the microtubule was enforced by demanding that all chain kink positions lie outside of the microtubule (yi2 + zi2) > (y02 + z02). Steric hindrance between antibody segments was implemented by restricting kink angles ϕi of sequential segments and by enforcing that the distance between any pair of points lying on two nonsequential segments was larger than dmin. Conformations that did not meet these criteria were neglected. The radial distance of the label from the microtubule center was determined from the y and z coordinates of the chain end point. Contour plots were created by 1.25 × 1.25 nm binning in y and z and subsequent 2d interpolation. For comparison with experimental cross-sections, the radial label density was revolved around the origin and convolved with the experimental (CRB plus residual drift) precisions in y and z. For determining the residuals, the convolved cross-section was rescaled (offset and magnitude) to best match the experimental cross-section (least-square fit), subtracted, and normalized by the maximum intensity of the rescaled cross-section.

Statistical Analysis

To investigate the potential significance of fitted labeling shell parameters (radius and thickness), we employed a two-sided F-test for pairwise comparisons by setting the standard deviations to times the 95% confidence intervals of fitted parameters, where n denotes the number of cross-sections that were pooled for the fit. p-values were calculated from F-values using the degrees of freedom for the numerator (=1) and denominator (=0.5(n1 + n2 – 2)). Comparable p-values were obtained by comparing the fit results of individual microtubule cross-sections (Supplementary Figure S3) for two conditions by an unpaired t-test.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.1c03677.

  • Experimental methods on mass spectrometry, SMLM setups, SMLM imaging, the determination of residual experimental errors, and circular dichroism spectroscopy measurements; Supplementary Figures S1 to S8; Supplementary Tables S1 to S6 (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Susanna M. Früh - Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;  Laboratory for MEMS Applications, IMTEK, Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany;  Orcidhttps://orcid.org/0000-0003-0313-9051
    • Ulf Matti - Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany;  Orcidhttps://orcid.org/0000-0001-5548-1081
    • Philipp R. Spycher - Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, 5232 Villigen, Switzerland
    • Marina Rubini - School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland;  Orcidhttps://orcid.org/0000-0002-3102-2558
    • Sebastian Lickert - Department of Health Sciences and Technology, ETH Zurich, 8093 Zurich, Switzerland
    • Thomas Schlichthaerle - Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany;  Max Planck Institute of Biochemistry, 82152 Martinsried, Germany;  Orcidhttps://orcid.org/0000-0003-4444-2575
    • Ralf Jungmann - Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany;  Max Planck Institute of Biochemistry, 82152 Martinsried, Germany;  Orcidhttps://orcid.org/0000-0003-4607-3312
    • Viola Vogel - Department of Health Sciences and Technology, ETH Zurich, 8093 Zurich, Switzerland
    • Jonas Ries - Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany;  Orcidhttps://orcid.org/0000-0002-6640-9250
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank the peer reviewers of this manuscript for their constructive critique. We would like to acknowledge R. Schibli for providing therapeutic antibodies. This work was supported by a fellowship of the Holcim Science Foundation (I.S.), the European Research Council (ERC CoG-724489, J.R.; ERC StG-680241, R.J.), the DFG (S.M.F., 397660978; R.J., JU 2957/1-1 and SFB 1032/A11), the AiF (S.M.F., 20436 N), the QBM graduate school (T.S.), the Center for Nano-Science (R.J.), the European Molecular Biology Laboratory (U.M. and J.R.), ETH Zurich (V.V.), the Max Planck Society/Foundation (J.R.), and RCSI (I.S.).

References

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    Agarwal, P.; Bertozzi, C. R. Site-Specific Antibody-Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development. Bioconjugate Chem. 2015, 26 (2), 176– 192,  DOI: 10.1021/bc5004982
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    11
    Site-Specific Antibody-Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development
    Agarwal, Paresh; Bertozzi, Carolyn R.
    Bioconjugate Chemistry (2015), 26 (2), 176-192CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A review. Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small mols. to create targeted drugs. Despite the simplicity of this concept, generation of clin. successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepd. by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixts. that cannot be further purified. With advances in the fields of bioorthogonal chem. and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chem. and protein engineering.
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  12. 12
    Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and Challenges for the Next Generation of Antibody–Drug Conjugates. Nat. Rev. Drug Discovery 2017, 16 (5), 315– 337,  DOI: 10.1038/nrd.2016.268
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    12
    Strategies and challenges for the next generation of antibody-drug conjugates
    Beck, Alain; Goetsch, Liliane; Dumontet, Charles; Corvaia, Nathalie
    Nature Reviews Drug Discovery (2017), 16 (5), 315-337CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. Antibody-drug conjugates (ADCs) are one of the fastest growing classes of oncol. therapeutics. After half a century of research, the approvals of brentuximab vedotin (in 2011) and trastuzumab emtansine (in 2013) have paved the way for ongoing clin. trials that are evaluating more than 60 further ADC candidates. The limited success of first-generation ADCs (developed in the early 2000s) informed strategies to bring second-generation ADCs to the market, which have higher levels of cytotoxic drug conjugation, lower levels of naked antibodies and more-stable linkers between the drug and the antibody. Furthermore, lessons learned during the past decade are now being used in the development of third-generation ADCs. In this Review, we discuss strategies to select the best target antigens as well as suitable cytotoxic drugs; the design of optimized linkers; the discovery of bioorthogonal conjugation chemistries; and toxicity issues. The selection and engineering of antibodies for site-specific drug conjugation, which will result in higher homogeneity and increased stability, as well as the quest for new conjugation chemistries and mechanisms of action, are priorities in ADC research.
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  13. 13
    Li, X.; Fang, T.; Boons, G. J. Preparation of Well-Defined Antibody-Drug Conjugates through Glycan Remodeling and Strain-Promoted Azide-Alkyne Cycloadditions. Angew. Chem., Int. Ed. 2014, 53 (28), 7179– 7182,  DOI: 10.1002/anie.201402606
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    13
    Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions
    Li, Xiuru; Fang, Tao; Boons, Geert-Jan
    Angewandte Chemie, International Edition (2014), 53 (28), 7179-7182CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chem. modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biol. systems and can be reacted by strain-promoted alkyne-azide cycloaddn. This method, which does not require genetic engineering, was used to produce an anti-CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.
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  14. 14
    Van Geel, R.; Wijdeven, M. A.; Heesbeen, R.; Verkade, J. M. M.; Wasiel, A. A.; Van Berkel, S. S.; Van Delft, F. L. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjugate Chem. 2015, 26 (11), 2233– 2242,  DOI: 10.1021/acs.bioconjchem.5b00224
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    14
    Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates
    van Geel, Remon; Wijdeven, Marloes A.; Heesbeen, Ryan; Verkade, Jorge M. M.; Wasiel, Anna A.; van Berkel, Sander S.; van Delft, Floris L.
    Bioconjugate Chemistry (2015), 26 (11), 2233-2242CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A robust, generally applicable, nongenetic technol. is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chem. The technol., termed GlycoConnect, is applicable to any IgG isotype irresp. of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.
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  15. 15
    Rosen, C. B.; Kodal, A. L. B.; Nielsen, J. S.; Schaffert, D. H.; Scavenius, C.; Okholm, A. H.; Voigt, N. V.; Enghild, J. J.; Kjems, J.; Tørring, T.; Gothelf, K. V. Template-Directed Covalent Conjugation of DNA to Native Antibodies, Transferrin and Other Metal-Binding Proteins. Nat. Chem. 2014, 6 (9), 804– 809,  DOI: 10.1038/nchem.2003
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    15
    Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins
    Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Joergen; Toerring, Thomas; Gothelf, Kurt V.
    Nature Chemistry (2014), 6 (9), 804-809CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    DNA-protein conjugates are important in bioanal. chem., mol. diagnostics and bionanotechnol., as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, prepg. such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and tech. challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the prodn. of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the const. domain.
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  16. 16
    Kline, T.; Steiner, A. R.; Penta, K.; Sato, A. K.; Hallam, T. J.; Yin, G. Methods to Make Homogenous Antibody Drug Conjugates. Pharm. Res. 2015, 32 (11), 3480– 3493,  DOI: 10.1007/s11095-014-1596-8
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    16
    Methods to Make Homogenous Antibody Drug Conjugates
    Kline, Toni; Steiner, Alexander R.; Penta, Kalyani; Sato, Aaron K.; Hallam, Trevor J.; Yin, Gang
    Pharmaceutical Research (2015), 32 (11), 3480-3493CODEN: PHREEB; ISSN:0724-8741. (Springer)
    Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small mol. chemistries that are currently used and being explored to develop specific, homogeneous ADCs.
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  17. 17
    Jeger, S.; Zimmermann, K.; Blanc, A.; Grünberg, J.; Honer, M.; Hunziker, P.; Struthers, H.; Schibli, R. Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase. Angew. Chem., Int. Ed. 2010, 49 (51), 9995– 9997,  DOI: 10.1002/anie.201004243
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    17
    Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase
    Jeger, Simone; Zimmermann, Kurt; Blanc, Alain; Gruenberg, Juergen; Honer, Michael; Hunziker, Peter; Struthers, Harriet; Schibli, Roger
    Angewandte Chemie, International Edition (2010), 49 (51), 9995-9997, S9995/1-S9995/46CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We have shown that enzymic modification of mAbs using bacterial transglutaminase (BTG) is site-specific and versatile (with the potential to be readily scaled up), and leads to homogeneous immunoconjugates with defined stoichiometries. The in vivo characteristics of such immunoconjugates are superior to those prepd. using chem. coupling methods. Since position 295 is located in the const. Fc region, the enzymic conjugation approach is applicable not only to other human IgG1s, but also to mAbs belonging to subtypes IgG2, IgG3, and IgG4, all of which conserve the Q295 residue. Thus, the method is broadly applicable and permits the systematic assessment and improvement of immunoconjugates.
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  18. 18
    Dennler, P.; Chiotellis, A.; Fischer, E.; Brégeon, D.; Belmant, C.; Gauthier, L.; Lhospice, F.; Romagne, F.; Schibli, R. Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody–Drug Conjugates. Bioconjugate Chem. 2014, 25 (3), 569– 578,  DOI: 10.1021/bc400574z
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    18
    Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates
    Dennler, Patrick; Chiotellis, Aristeidis; Fischer, Eliane; Bregeon, Delphine; Belmant, Christian; Gauthier, Laurent; Lhospice, Florence; Romagne, Francois; Schibli, Roger
    Bioconjugate Chemistry (2014), 25 (3), 569-578CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Most chem. techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixt. of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-deriv. To this aim, we investigated two different chem. approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddn. (SPAAC). Direct enzymic attachment of MMAE-spacer derivs. at an 80 M excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymic approach only required a 2.5 M excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, resp. Thus, the chemo-enzymic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.
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    Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126 (46), 15046– 15047,  DOI: 10.1021/ja044996f
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    19
    A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems
    Agard, Nicholas J.; Prescher, Jennifer A.; Bertozzi, Carolyn R.
    Journal of the American Chemical Society (2004), 126 (46), 15046-15047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Selective chem. reactions that are orthogonal to the diverse functionality of biol. systems have become important tools in the field of chem. biol. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3+2] cycloaddn. of azides and alkynes ("click chem."). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidn. and synthetic challenges. Click chem. obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3+2] cycloaddn. between cyclooctynes and azides that proceeds under physiol. conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomols. in vitro and on living cells, with no apparent toxicity.
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  20. 20
    Helmerich, D. A.; Beliu, G.; Sauer, M. Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer. ACS Nano 2020, 14 (10), 12629– 12641,  DOI: 10.1021/acsnano.0c06099
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    20
    Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer
    Helmerich Dominic A; Beliu Gerti; Sauer Markus
    ACS nano (2020), 14 (10), 12629-12641 ISSN:.
    The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1-8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.
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  21. 21
    Pleiner, T.; Bates, M.; Görlich, D. A Toolbox of Anti-Mouse and Anti-Rabbit IgG Secondary Nanobodies. J. Cell Biol. 2018, 217 (3), 1143– 1154,  DOI: 10.1083/jcb.201709115
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    21
    A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies
    Pleiner, Tino; Bates, Mark; Goerlich, Dirk
    Journal of Cell Biology (2018), 217 (3), 1143-1159CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
    Polyclonal anti-IgG (anti-IgG) secondary antibodies are essential tools for many mol. biol. techniques and diagnostic tests. Their animal-based prodn. is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody prodn. in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chem. for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresoln. microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.
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    Vaughan, J. C.; Dempsey, G. T.; Sun, E.; Zhuang, X. Phosphine-Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy. J. Am. Chem. Soc. 2013, 135, 1197– 1200,  DOI: 10.1021/ja3105279
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    22
    Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy
    Vaughan, Joshua C.; Dempsey, Graham T.; Sun, Eileen; Zhuang, Xiaowei
    Journal of the American Chemical Society (2013), 135 (4), 1197-1200CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The authors report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, the quenching reaction occurs by 1,4-addn. of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissocs. the adduct and returns the dye to the fluorescent state. TCEP quenching can be used for super-resoln. imaging as well as for other applications, such as differentiating between mols. inside and outside the cell.
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    Olivier, N.; Keller, D.; Gönczy, P.; Manley, S. Resolution Doubling in 3D-STORM Imaging through Improved Buffers. PLoS One 2013, 8 (7), 1– 9,  DOI: 10.1371/journal.pone.0069004
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    Endesfelder, U.; Malkusch, S.; Fricke, F.; Heilemann, M. A Simple Method to Estimate the Average Localization Precision of a Single-Molecule Localization Microscopy Experiment. Histochem. Cell Biol. 2014, 141 (6), 629– 638,  DOI: 10.1007/s00418-014-1192-3
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    24
    A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment
    Endesfelder, Ulrike; Malkusch, Sebastian; Fricke, Franziska; Heilemann, Mike
    Histochemistry and Cell Biology (2014), 141 (6), 629-638CODEN: HCBIFP; ISSN:0948-6143. (Springer)
    The localization precision is a crucial and important parameter for single-mol. localization microscopy (SMLM) and directly influences the achievable spatial resoln. It primarily depends on exptl. imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to est. the av. exptl. localization precision in SMLM, based on the nearest neighbor anal. By exploring different exptl. and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σSMLM are obtained. Knowing σSMLM is a prerequisite for consistent visualization or any quant. structural anal., e.g., cluster anal. or colocalization studies.
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    Li, Y.; Mund, M.; Hoess, P.; Deschamps, J.; Matti, U.; Nijmeijer, B.; Sabinina, V. J.; Ellenberg, J.; Schoen, I.; Ries, J. Real-Time 3D Single-Molecule Localization Using Experimental Point Spread Functions. Nat. Methods 2018, 15 (5), 367– 369,  DOI: 10.1038/nmeth.4661
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    25
    Real-time 3D single-molecule localization using experimental point spread functions
    Li, Yiming; Mund, Markus; Hoess, Philipp; Deschamps, Joran; Matti, Ulf; Nijmeijer, Bianca; Sabinina, Vilma Jimenez; Ellenberg, Jan; Schoen, Ingmar; Ries, Jonas
    Nature Methods (2018), 15 (5), 367-369CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    We present a real-time fitter for 3D single-mol. localization microscopy using exptl. point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resoln. microscopy broadly accessible, even on std. microscopes without dedicated 3D optics.
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    Auer, A.; Schlichthaerle, T.; Woehrstein, J. B.; Schueder, F.; Strauss, M. T.; Grabmayr, H.; Jungmann, R. Nanometer-Scale Multiplexed Super-Resolution Imaging with an Economic 3D-DNA-PAINT Microscope. ChemPhysChem 2018, 19 (22), 3024– 3034,  DOI: 10.1002/cphc.201800630
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    26
    Nanometer-scale Multiplexed Super-Resolution Imaging with an Economic 3D-DNA-PAINT Microscope
    Auer, Alexander; Schlichthaerle, Thomas; Woehrstein, Johannes B.; Schueder, Florian; Strauss, Maximilian T.; Grabmayr, Heinrich; Jungmann, Ralf
    ChemPhysChem (2018), 19 (22), 3024-3034CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Optical super-resoln. microscopy is rapidly changing the way imaging studies in the biol. and biomedical sciences are conducted. Due to the unique capability of achieving mol. contrast using fluorescent labels and sub-diffraction resoln. down to a few tens of nanometers, super-resoln. is developing as an attractive imaging modality. While the increased spatial resoln. has already enabled structural studies at unprecedented mol. detail, the wide-spread use of super-resoln. approaches as a std. characterization technique in biol. labs. has thus far been prevented by mainly two issues: (1) Intricate sample prepn. and image acquisition and (2) costly and complex instrumentation. We here introduce a combination of the recently developed super-resoln. technique DNA-PAINT (DNA points accumulation for imaging in nanoscale topog.) with an easy-to-replicate, custom-built 3D single-mol. microscope (termed liteTIRF) that is an order of magnitude more economic in cost compared to most com. systems. We assay the performance of our system using synthetic two- and three-dimensional DNA origami structures and show the applicability to single- and multiplexed cellular imaging.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVGltLzM&md5=de5d6cc8edf56158bd04e01fee92015b
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    Journal of Molecular Biology (2017), 429 (5), 633-646CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    A no. of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtained high-resoln. (3.9-4.2 Å) cryo-electron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on β-tubulin. We find that each mol. has unique distinct structural effects on the MT lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the "seam" of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e., non-seam) contacts by which it regularizes the MT lattice. In contrast, binding of either Taxol or zampanolide induces MT heterogeneity. In doubly bound MTs, peloruside overrides the heterogeneity induced by Taxol binding. Our structural anal. illustrates distinct mechanisms of these drugs for stabilizing the MT lattice and is of relevance to the possible use of combinations of MSAs to regulate MT activity and improve therapeutic potential.
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    Biophysical Journal (2010), 99 (3), 905-913CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    At 150 kDa, antibodies of the IgG class are too large for their structure to be detd. with current NMR methodologies. Because of hinge-region flexibility, it is difficult to obtain at.-level structural information from the crystal, and questions regarding antibody structure and dynamics in soln. remain unaddressed. Here we describe the construction of a model of a human IgG1 monoclonal antibody (trastuzumab) from the crystal structures of fragments. We use a combination of mol.-dynamics (MD) simulation, continuum hydrodynamics modeling, and exptl. diffusion measurements to explore antibody behavior in aq. soln. Hydrodynamic modeling provides a link between the at.-level details of MD simulation and the size- and shape-dependent data provided by hydrodynamic measurements. Eight independent 40 ns MD trajectories were obtained with the AMBER program suite. The ensemble av. of the computed transport properties over all of the MD trajectories agrees remarkably well with the value of the translational diffusion coeff. obtained with dynamic light scattering at 20°C and 27°C, and the intrinsic viscosity measured at 20°C. Therefore, our MD results likely represent a realistic sampling of the conformational space that an antibody explores in aq. soln.
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    Biochemistry (1997), 36 (7), 1581-1597CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was detd. by mol. replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 Å resoln. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron d. maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relation. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to const. domains. Variable domains exhibit no symmetry relation as a consequence of the 16° difference in Fab elbow angles. Variable domain pair assocns. VL:VH for the Fabs are virtually the same, and corresponding CDRs of the 2 Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by x-ray anal. of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial mol. replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.
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    The issue of protein dynamics and its implications in the biol. function of proteins are arousing greater and greater interest in different fields of mol. biol. In cryo-electron tomog. expts. one may take several snapshots of a given biol. macromol. In principle, a large enough collection of snapshots of the mol. may then be used to calc. its equil. configuration in terms of the exptl. accessible degrees of freedom and, hence, to est. its potential energy. This information would be crucial in order to analyze the biol. functions of biomols. by directly accessing the relevant dynamical indicators. In this article, we analyze the results of cryo-electron tomog. expts. performed on monoclonal murine IgG2a antibodies. We measure the equil. distribution of the mol. in terms of the relevant angular coordinates and build a mech. model of the antibody dynamics. This approach enables us to derive an explicit expression of the IgG potential energy. Furthermore, we discuss the configuration space at equil. in relation to results from other techniques, and we set our discussion in the context of the current debate regarding conformation and flexibility of antibodies.
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    Detn. of the mol. architecture of synapses requires nanoscopic image resoln. and specific mol. recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresoln. fluorescence imaging method to visualize the mol. architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quant. anal. of a large no. of synapses from different brain regions. To demonstrate the capabilities of this approach, we have detd. the organization of ten protein components of the presynaptic active zone and the postsynaptic d. Variations in synapse morphol., neurotransmitter receptor compn., and receptor distribution were obsd. both among synapses and across different brain regions. Combination with optogenetics further allowed mol. events assocd. with synaptic plasticity to be resolved at the single-synapse level.
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    Molecular immunology (2000), 37 (12-13), 697-706 ISSN:0161-5890.
    Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.
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    Stability of therapeutic IgG prepns. is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concn. (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amt. of additives without endangering quality and stability. Since both biol. function and aggregation depend on the conformational properties of individual IgG mols., two sensitive and rapid phys. methods were introduced to assess conformational changes and structural stability as a function of pH and addn. of std. stabilizers. It was obsd. that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiol. acceptable concn. have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concn. as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of arom. side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temp. are decisive factors for the long-term stability of IgG solns. An increase in the dimer content was obsd. upon storage at 5°C which was partly reverted upon incubation at 37°C. Storage at temps. higher than 5°C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concn., it is advisable to omit sugars at i.v. Ig (IVIG) formulation. Hydrophobic amino acids give promising alternatives.
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    Biophysical Journal (2000), 78 (1), 394-404CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The denaturation of IgG was studied by different calorimetric methods and CD spectroscopy. The thermogram of the Ig showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temp. and pH. The two peaks represent the Fab and Fc fragments of the IgG mol. The Fab fragment is most sensitive to heat treatment, whereas the Fc fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temp., the unfolding is the rate-detg. step in the overall denaturation process. At higher temps. where a relatively high concn. of (partially) unfolded IgG mols. is present, the rate of aggregation is so fast that IgG mols. become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The CD spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as obsd. by calorimetry and the changes in secondary structure derived from CD. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.
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    Nature Methods (2020), 17 (9), 870-872CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    There is no expanded citation for this reference.
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  • Abstract

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    Scheme 1

    Scheme 1. Site-specific labeling of IgG antibodies. In a first reaction, N-linked glycans are removed by PNGaseF, and only the available glutamines (Q) at position −2 relative to the deglycosylated sites are modified with H2N-PEG3-N3 catalyzed by microbial transglutaminase (mTG). In a second reaction, fluorophores(-DIBO) or ssDNA(-DBCO) are attached to the azide-modified antibodies using strain-promoted azide–alkyne cycloaddition (SPAAC).
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    Figure 1

    Figure 1. Sequence alignment and evaluation of potential modification sites for different IgG subclasses from different host species. Sequences were sourced from the UniProt database and aligned with respect to the N-linked glycosylation sites (gray background). The glutamine at position −2 (black background) is preserved across the majority of IgG subtypes and species for which sequence information was available. *Potential conflict with additional glutamine marked in red; Fl = Florida.

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    Figure 2

    Figure 2. 3D SMLM imaging of microtubule networks and determination of labeling shell dimensions for indirect and direct immunolabeling. (a–d) 3D STORM using Alexa Fluor 647 labeled antibodies in fixed U2OS cells. (a) Schematic (not to scale). (b) Indirect immunolabeling using a randomly labeled secondary donkey-anti-mouse (NHS 2°). (c) Indirect immunolabeling using a site-specifically-labeled secondary donkey-anti-mouse (2°). (d) Direct immunostaining using a site-specifically-labeled primary mouse-anti-α-tubulin (1°). (e–h) 3D DNA-PAINT using DNA-labeled antibodies in U2OS cells (f) and human platelets (g, h). (e) Schematic (not to scale). (f, g) Indirect immunolabeling using a site-specifically-labeled donkey-anti-mouse secondary (2°). (h) Direct labeling using a site-specifically-labeled primary mouse-anti-α-tubulin (1°). For all conditions, representative 3D SMLM images are shown (left). Labeling shell dimensions around microtubules were determined from averaged experimental yz cross-sections and fitted label distributions (right). The fitted label distribution was a Gaussian ring kernel (r: radius; w: full width at half-maximum) convolved with the localization precisions in y and z, respectively. (i) Comparison of center positions and widths of labeling shell dimensions for the different labeling strategies in b–d and f–h.

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    Figure 3

    Figure 3. Monte Carlo simulations of antibody conformations at microtubules. (a) Location of the epitope of the anti-α-tubulin antibody in our geometric model (right) based on the cryo-EM structure of a microtubule (left; PDB 5SYF, ref (27); molecular surface rendered using Mol*, ref (33)). (b) Each IgG molecule was modeled by two segments corresponding to Fab and Fc fragments connected by a flexible hinge region. Right: Parametrization of Fab, Fc, and linker segments of the labeled primary antibody. Left: Dimensions were based on the crystallographic structure of IgG2a (PDB 1IGT, ref (30), visualization by Mol*). While the location of the modification is precisely known (Glu), the binding sites of polyclonal secondary antibodies are assumed to be evenly distributed over the Fc region. For a schematic of indirect immunolabeling, see Supplementary Figure S5c). Simulated reporter distribution for primary antibodies. Left: yz cross-section. Right: Radial distribution. (d) Simulated reporter distribution for primary plus secondary antibody complexes. Panels as in (c). (e) Comparison of reporter distributions for primary antibodies between simulations (left; convolved with the localization and experimental imprecision) and experiments (middle: pooled cross-sections as in Figure 2). Right: Normalized residuals of the difference between experiment and model. Top: STORM. Bottom: DNA-PAINT. (f) Comparison of reporter distributions for secondary antibodies between simulations and experiments. Panels as in (d).

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      Microtubules are hollow biopolymers of 25-nm diam. and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resoln. microscopy techniques can detect specific structures at an increased resoln., but the narrow spacing between neuronal microtubules poses challenges because most existing labeling strategies increase the effective microtubule diam. by 20-40 nm and will thereby blend neighboring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resoln. imaging of microtubules with a decreased apparent diam. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiol.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xmt1Gksbs%253D&md5=678f32bb3f032e786fb8ca42d893030a
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      This article shows that aptamers, which are single-stranded DNA or RNA oligonucleotides, are potential probes that would complement nanobodies for super-resoln. imaging. Three aptamers were tested for mols. involved in endosomal trafficking: the transferrin receptor (TfnR; aptamer size ∼15 kDa), the prostate-specific membrane antigen (PSMA; ∼15 kDa) and the epidermal growth factor receptor (EGFR; ∼36 kDa). Epitope recognition with aptamers was higher than with conventional antibodies, and this allowed the aptamers to provide superior super-resoln. images, just as the nanobodies did, when investigated using stimulated emission depletion (STED) microscopy. The high epitope recognition also permitted rapid live super-resoln. imaging.
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      DNA-protein conjugates are important in bioanal. chem., mol. diagnostics and bionanotechnol., as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, prepg. such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and tech. challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the prodn. of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the const. domain.
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      16
      Methods to Make Homogenous Antibody Drug Conjugates
      Kline, Toni; Steiner, Alexander R.; Penta, Kalyani; Sato, Aaron K.; Hallam, Trevor J.; Yin, Gang
      Pharmaceutical Research (2015), 32 (11), 3480-3493CODEN: PHREEB; ISSN:0724-8741. (Springer)
      Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small mol. chemistries that are currently used and being explored to develop specific, homogeneous ADCs.
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    17. 17
      Jeger, S.; Zimmermann, K.; Blanc, A.; Grünberg, J.; Honer, M.; Hunziker, P.; Struthers, H.; Schibli, R. Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase. Angew. Chem., Int. Ed. 2010, 49 (51), 9995– 9997,  DOI: 10.1002/anie.201004243
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      17
      Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase
      Jeger, Simone; Zimmermann, Kurt; Blanc, Alain; Gruenberg, Juergen; Honer, Michael; Hunziker, Peter; Struthers, Harriet; Schibli, Roger
      Angewandte Chemie, International Edition (2010), 49 (51), 9995-9997, S9995/1-S9995/46CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have shown that enzymic modification of mAbs using bacterial transglutaminase (BTG) is site-specific and versatile (with the potential to be readily scaled up), and leads to homogeneous immunoconjugates with defined stoichiometries. The in vivo characteristics of such immunoconjugates are superior to those prepd. using chem. coupling methods. Since position 295 is located in the const. Fc region, the enzymic conjugation approach is applicable not only to other human IgG1s, but also to mAbs belonging to subtypes IgG2, IgG3, and IgG4, all of which conserve the Q295 residue. Thus, the method is broadly applicable and permits the systematic assessment and improvement of immunoconjugates.
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    18. 18
      Dennler, P.; Chiotellis, A.; Fischer, E.; Brégeon, D.; Belmant, C.; Gauthier, L.; Lhospice, F.; Romagne, F.; Schibli, R. Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody–Drug Conjugates. Bioconjugate Chem. 2014, 25 (3), 569– 578,  DOI: 10.1021/bc400574z
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      18
      Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates
      Dennler, Patrick; Chiotellis, Aristeidis; Fischer, Eliane; Bregeon, Delphine; Belmant, Christian; Gauthier, Laurent; Lhospice, Florence; Romagne, Francois; Schibli, Roger
      Bioconjugate Chemistry (2014), 25 (3), 569-578CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Most chem. techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixt. of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-deriv. To this aim, we investigated two different chem. approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddn. (SPAAC). Direct enzymic attachment of MMAE-spacer derivs. at an 80 M excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymic approach only required a 2.5 M excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, resp. Thus, the chemo-enzymic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.
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    19. 19
      Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126 (46), 15046– 15047,  DOI: 10.1021/ja044996f
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      19
      A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems
      Agard, Nicholas J.; Prescher, Jennifer A.; Bertozzi, Carolyn R.
      Journal of the American Chemical Society (2004), 126 (46), 15046-15047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Selective chem. reactions that are orthogonal to the diverse functionality of biol. systems have become important tools in the field of chem. biol. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3+2] cycloaddn. of azides and alkynes ("click chem."). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidn. and synthetic challenges. Click chem. obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3+2] cycloaddn. between cyclooctynes and azides that proceeds under physiol. conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomols. in vitro and on living cells, with no apparent toxicity.
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    20. 20
      Helmerich, D. A.; Beliu, G.; Sauer, M. Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer. ACS Nano 2020, 14 (10), 12629– 12641,  DOI: 10.1021/acsnano.0c06099
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      20
      Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer
      Helmerich Dominic A; Beliu Gerti; Sauer Markus
      ACS nano (2020), 14 (10), 12629-12641 ISSN:.
      The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1-8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.
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    21. 21
      Pleiner, T.; Bates, M.; Görlich, D. A Toolbox of Anti-Mouse and Anti-Rabbit IgG Secondary Nanobodies. J. Cell Biol. 2018, 217 (3), 1143– 1154,  DOI: 10.1083/jcb.201709115
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      21
      A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies
      Pleiner, Tino; Bates, Mark; Goerlich, Dirk
      Journal of Cell Biology (2018), 217 (3), 1143-1159CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      Polyclonal anti-IgG (anti-IgG) secondary antibodies are essential tools for many mol. biol. techniques and diagnostic tests. Their animal-based prodn. is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody prodn. in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chem. for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresoln. microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.
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    22. 22
      Vaughan, J. C.; Dempsey, G. T.; Sun, E.; Zhuang, X. Phosphine-Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy. J. Am. Chem. Soc. 2013, 135, 1197– 1200,  DOI: 10.1021/ja3105279
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      22
      Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy
      Vaughan, Joshua C.; Dempsey, Graham T.; Sun, Eileen; Zhuang, Xiaowei
      Journal of the American Chemical Society (2013), 135 (4), 1197-1200CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, the quenching reaction occurs by 1,4-addn. of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissocs. the adduct and returns the dye to the fluorescent state. TCEP quenching can be used for super-resoln. imaging as well as for other applications, such as differentiating between mols. inside and outside the cell.
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    23. 23
      Olivier, N.; Keller, D.; Gönczy, P.; Manley, S. Resolution Doubling in 3D-STORM Imaging through Improved Buffers. PLoS One 2013, 8 (7), 1– 9,  DOI: 10.1371/journal.pone.0069004
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    24. 24
      Endesfelder, U.; Malkusch, S.; Fricke, F.; Heilemann, M. A Simple Method to Estimate the Average Localization Precision of a Single-Molecule Localization Microscopy Experiment. Histochem. Cell Biol. 2014, 141 (6), 629– 638,  DOI: 10.1007/s00418-014-1192-3
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      24
      A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment
      Endesfelder, Ulrike; Malkusch, Sebastian; Fricke, Franziska; Heilemann, Mike
      Histochemistry and Cell Biology (2014), 141 (6), 629-638CODEN: HCBIFP; ISSN:0948-6143. (Springer)
      The localization precision is a crucial and important parameter for single-mol. localization microscopy (SMLM) and directly influences the achievable spatial resoln. It primarily depends on exptl. imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to est. the av. exptl. localization precision in SMLM, based on the nearest neighbor anal. By exploring different exptl. and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σSMLM are obtained. Knowing σSMLM is a prerequisite for consistent visualization or any quant. structural anal., e.g., cluster anal. or colocalization studies.
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    25. 25
      Li, Y.; Mund, M.; Hoess, P.; Deschamps, J.; Matti, U.; Nijmeijer, B.; Sabinina, V. J.; Ellenberg, J.; Schoen, I.; Ries, J. Real-Time 3D Single-Molecule Localization Using Experimental Point Spread Functions. Nat. Methods 2018, 15 (5), 367– 369,  DOI: 10.1038/nmeth.4661
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      25
      Real-time 3D single-molecule localization using experimental point spread functions
      Li, Yiming; Mund, Markus; Hoess, Philipp; Deschamps, Joran; Matti, Ulf; Nijmeijer, Bianca; Sabinina, Vilma Jimenez; Ellenberg, Jan; Schoen, Ingmar; Ries, Jonas
      Nature Methods (2018), 15 (5), 367-369CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      We present a real-time fitter for 3D single-mol. localization microscopy using exptl. point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resoln. microscopy broadly accessible, even on std. microscopes without dedicated 3D optics.
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    26. 26
      Auer, A.; Schlichthaerle, T.; Woehrstein, J. B.; Schueder, F.; Strauss, M. T.; Grabmayr, H.; Jungmann, R. Nanometer-Scale Multiplexed Super-Resolution Imaging with an Economic 3D-DNA-PAINT Microscope. ChemPhysChem 2018, 19 (22), 3024– 3034,  DOI: 10.1002/cphc.201800630
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      26
      Nanometer-scale Multiplexed Super-Resolution Imaging with an Economic 3D-DNA-PAINT Microscope
      Auer, Alexander; Schlichthaerle, Thomas; Woehrstein, Johannes B.; Schueder, Florian; Strauss, Maximilian T.; Grabmayr, Heinrich; Jungmann, Ralf
      ChemPhysChem (2018), 19 (22), 3024-3034CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Optical super-resoln. microscopy is rapidly changing the way imaging studies in the biol. and biomedical sciences are conducted. Due to the unique capability of achieving mol. contrast using fluorescent labels and sub-diffraction resoln. down to a few tens of nanometers, super-resoln. is developing as an attractive imaging modality. While the increased spatial resoln. has already enabled structural studies at unprecedented mol. detail, the wide-spread use of super-resoln. approaches as a std. characterization technique in biol. labs. has thus far been prevented by mainly two issues: (1) Intricate sample prepn. and image acquisition and (2) costly and complex instrumentation. We here introduce a combination of the recently developed super-resoln. technique DNA-PAINT (DNA points accumulation for imaging in nanoscale topog.) with an easy-to-replicate, custom-built 3D single-mol. microscope (termed liteTIRF) that is an order of magnitude more economic in cost compared to most com. systems. We assay the performance of our system using synthetic two- and three-dimensional DNA origami structures and show the applicability to single- and multiplexed cellular imaging.
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    27. 27
      Kellogg, E. H.; Hejab, N. M. A.; Howes, S.; Northcote, P.; Miller, J. H.; Díaz, J. F.; Downing, K. H.; Nogales, E. Insights into the Distinct Mechanisms of Action of Taxane and Non-Taxane Microtubule Stabilizers from Cryo-EM Structures. J. Mol. Biol. 2017, 429 (5), 633– 646,  DOI: 10.1016/j.jmb.2017.01.001
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      27
      Insights into the Distinct Mechanisms of Action of Taxane and Non-Taxane Microtubule Stabilizers from Cryo-EM Structures
      Kellogg, Elizabeth H.; Hejab, Nisreen M. A.; Howes, Stuart; Northcote, Peter; Miller, John H.; Diaz, J. Fernando; Downing, Kenneth H.; Nogales, Eva
      Journal of Molecular Biology (2017), 429 (5), 633-646CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      A no. of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtained high-resoln. (3.9-4.2 Å) cryo-electron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on β-tubulin. We find that each mol. has unique distinct structural effects on the MT lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the "seam" of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e., non-seam) contacts by which it regularizes the MT lattice. In contrast, binding of either Taxol or zampanolide induces MT heterogeneity. In doubly bound MTs, peloruside overrides the heterogeneity induced by Taxol binding. Our structural anal. illustrates distinct mechanisms of these drugs for stabilizing the MT lattice and is of relevance to the possible use of combinations of MSAs to regulate MT activity and improve therapeutic potential.
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    28. 28
      Brandt, J. P.; Patapoff, T. W.; Aragon, S. R. Construction, MD Simulation, and Hydrodynamic Validation of an All-Atom Model of a Monoclonal IgG Antibody. Biophys. J. 2010, 99 (3), 905– 913,  DOI: 10.1016/j.bpj.2010.05.003
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      28
      Construction, MD Simulation, and Hydrodynamic Validation of an All-Atom Model of a Monoclonal IgG Antibody
      Brandt, J. Paul; Patapoff, Thomas W.; Aragon, Sergio R.
      Biophysical Journal (2010), 99 (3), 905-913CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      At 150 kDa, antibodies of the IgG class are too large for their structure to be detd. with current NMR methodologies. Because of hinge-region flexibility, it is difficult to obtain at.-level structural information from the crystal, and questions regarding antibody structure and dynamics in soln. remain unaddressed. Here we describe the construction of a model of a human IgG1 monoclonal antibody (trastuzumab) from the crystal structures of fragments. We use a combination of mol.-dynamics (MD) simulation, continuum hydrodynamics modeling, and exptl. diffusion measurements to explore antibody behavior in aq. soln. Hydrodynamic modeling provides a link between the at.-level details of MD simulation and the size- and shape-dependent data provided by hydrodynamic measurements. Eight independent 40 ns MD trajectories were obtained with the AMBER program suite. The ensemble av. of the computed transport properties over all of the MD trajectories agrees remarkably well with the value of the translational diffusion coeff. obtained with dynamic light scattering at 20°C and 27°C, and the intrinsic viscosity measured at 20°C. Therefore, our MD results likely represent a realistic sampling of the conformational space that an antibody explores in aq. soln.
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    29. 29
      De Michele, C.; De Los Rios, P.; Foffi, G.; Piazza, F. Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces. PLoS Comput. Biol. 2016, 12 (3), 1– 17,  DOI: 10.1371/journal.pcbi.1004752
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    30. 30
      Harris, L. J.; Larson, S. B.; Hasel, K. W.; McPherson, A. Refined Structure of an Intact IgG2a Monoclonal Antibody. Biochemistry 1997, 36 (7), 1581– 1597,  DOI: 10.1021/bi962514+
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      30
      Refined Structure of an Intact IgG2a Monoclonal Antibody
      Harris, Lisa J.; Larson, Steven B.; Hasel, Karl W.; McPherson, Alexander
      Biochemistry (1997), 36 (7), 1581-1597CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was detd. by mol. replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 Å resoln. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron d. maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relation. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to const. domains. Variable domains exhibit no symmetry relation as a consequence of the 16° difference in Fab elbow angles. Variable domain pair assocns. VL:VH for the Fabs are virtually the same, and corresponding CDRs of the 2 Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by x-ray anal. of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial mol. replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.
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    31. 31
      Bongini, L.; Fanelli, D.; Piazza, F.; De Los Rios, P.; Sandin, S.; Skoglund, U. Freezing Immunoglobulins to See Them Move. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (17), 6466– 6471,  DOI: 10.1073/pnas.0400119101
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      31
      Freezing immunoglobulins to see them move
      Bongini, L.; Fanelli, D.; Piazza, F.; De Los Rios, P.; Sandin, S.; Skoglund, U.
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (17), 6466-6471CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The issue of protein dynamics and its implications in the biol. function of proteins are arousing greater and greater interest in different fields of mol. biol. In cryo-electron tomog. expts. one may take several snapshots of a given biol. macromol. In principle, a large enough collection of snapshots of the mol. may then be used to calc. its equil. configuration in terms of the exptl. accessible degrees of freedom and, hence, to est. its potential energy. This information would be crucial in order to analyze the biol. functions of biomols. by directly accessing the relevant dynamical indicators. In this article, we analyze the results of cryo-electron tomog. expts. performed on monoclonal murine IgG2a antibodies. We measure the equil. distribution of the mol. in terms of the relevant angular coordinates and build a mech. model of the antibody dynamics. This approach enables us to derive an explicit expression of the IgG potential energy. Furthermore, we discuss the configuration space at equil. in relation to results from other techniques, and we set our discussion in the context of the current debate regarding conformation and flexibility of antibodies.
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    32. 32
      Dani, A.; Huang, B.; Bergan, J.; Dulac, C.; Zhuang, X. Superresolution Imaging of Chemical Synapses in the Brain. Neuron 2010, 68 (5), 843– 856,  DOI: 10.1016/j.neuron.2010.11.021
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      32
      Superresolution Imaging of Chemical Synapses in the Brain
      Dani, Adish; Huang, Bo; Bergan, Joseph; Dulac, Catherine; Zhuang, Xiao-Wei
      Neuron (2010), 68 (5), 843-856CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      Detn. of the mol. architecture of synapses requires nanoscopic image resoln. and specific mol. recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresoln. fluorescence imaging method to visualize the mol. architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quant. anal. of a large no. of synapses from different brain regions. To demonstrate the capabilities of this approach, we have detd. the organization of ten protein components of the presynaptic active zone and the postsynaptic d. Variations in synapse morphol., neurotransmitter receptor compn., and receptor distribution were obsd. both among synapses and across different brain regions. Combination with optogenetics further allowed mol. events assocd. with synaptic plasticity to be resolved at the single-synapse level.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFGhu73I&md5=07c554f9890e13d6767bc4205c3001f6
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      Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH2 domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.
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      Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.
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      The denaturation of IgG was studied by different calorimetric methods and CD spectroscopy. The thermogram of the Ig showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temp. and pH. The two peaks represent the Fab and Fc fragments of the IgG mol. The Fab fragment is most sensitive to heat treatment, whereas the Fc fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temp., the unfolding is the rate-detg. step in the overall denaturation process. At higher temps. where a relatively high concn. of (partially) unfolded IgG mols. is present, the rate of aggregation is so fast that IgG mols. become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The CD spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as obsd. by calorimetry and the changes in secondary structure derived from CD. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.
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      SMAP: a modular super-resolution microscopy analysis platform for SMLM data
      Ries, Jonas
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      There is no expanded citation for this reference.
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    • Experimental methods on mass spectrometry, SMLM setups, SMLM imaging, the determination of residual experimental errors, and circular dichroism spectroscopy measurements; Supplementary Figures S1 to S8; Supplementary Tables S1 to S6 (PDF)


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