Fast antibody fragment motion: flexible linkers act as entropic spring

Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, D.

doi:10.1038/srep22148

Cited by 32 publications

(43 citation statements)

References 62 publications

(87 reference statements)

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“…The obtained D t (w t ) was found to agree quantitatively with the theory of colloidal hard-sphere suspensions if an empirical effective hydrodynamic radius R eff = 1.4((3/(4π))V p ) 1/3 is used to account for anisotropy of the protein structure. This value may also reflect the presence of a significant motion of the three branches of the protein, as recently suggested by a NSE study on the internal dynamics of IgG by Stingaciu et al (2016) and an earlier study based on a comparison between crystal structures (Saphire et al, 2002). Moreover, the radius R eff may effectively account for the anisotropy of the structure of IgG, which is not considered in the theory for hard-spheres.…”

Section: Global Diffusionmentioning

confidence: 83%

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Dynamics of proteins in solution

Grimaldo

Roosen-Runge

Zhang

et al. 2019

Quart. Rev. Biophys.

104

164

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The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time- and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.

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Section: Global Diffusionmentioning

confidence: 83%

“…This approach accounts for the full anisotropy of the shape and diffusion, but depends on an available protein structure, which furthermore is assumed to be rigid. From the diffusion tensor D, the apparent collective diffusion coefficient D (c) app (q) is given by (Biehl et al, 2011;Stingaciu et al, 2016)…”

Section: Diffusion Of the Entire Proteinmentioning

confidence: 99%

Dynamics of proteins in solution

Grimaldo

Roosen-Runge

Zhang

et al. 2019

Quart. Rev. Biophys.

104

164

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“…Prediction of the anisotropy of the bound fraction using model calculations established that the anisotropy vs. DOL curve measured for antibody stocks can be used as a calibration curve for determining the mean DOL of the bound fraction. The decreased affinity may be the consequence of the altered or inhibited internal flexibility of antibodies required for antigen binding (43,44). Although antibodies and fluorophores show remarkable difference regarding their sensitivity to fluorescence labeling, the mean DOL of the bound fraction is almost always lower than that of the stock, albeit the DOL-dependence of the fluorescence intensity of the stock and of the bound fraction differ from each other.…”

Section: Discussionmentioning

confidence: 99%

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Szabó

Szendi-Szatmári

Ujlaky-Nagy

et al. 2018

Biophysical Journal

107

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Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

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“…Our IgG1 and IgG4 studies complement other recent investigations on antibody solution structures. A neutron spin echo study (55) was conducted with a heterogeneous mixture of polyclonal monomeric and dimeric human IgG from plasma, comprised of the four IgG1, IgG2, IgG3, and IgG4 subclasses with different hinge structures between the subclasses. Similar to earlier studies of antibody flexibility involving the Fab and Fc regions, flexibility was detected as contributions to translational and rotational diffusion motions between the Fab and Fc regions.…”

Section: Discussionmentioning

confidence: 99%

Atomistic Modeling of Scattering Curves for Human IgG1/4 Reveals New Structure-Function Insights

Wright

Elliston

Hui

et al. 2019

Biophysical Journal

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Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit R factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcgRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4.

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Fast antibody fragment motion: flexible linkers act as entropic spring

Cited by 32 publications

References 62 publications

Dynamics of proteins in solution

Dynamics of proteins in solution

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Atomistic Modeling of Scattering Curves for Human IgG1/4 Reveals New Structure-Function Insights

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