Quantitative Analysis of Protein Self‐Association by Sedimentation Velocity

Abstract

Sedimentation velocity analytical ultracentrifugation is a powerful classical method to study protein self‐association processes in solution based on the size‐dependent macromolecular migration in the centrifugal field. This technique can elucidate the assembly scheme, measure affinities ranging from picomolar to millimolar Kd, and in favorable cases provide information on oligomer lifetimes and hydrodynamic shape. The present step‐by‐step protocols detail the essential steps of instrument calibration, experimental setup, and data analysis. Using a widely available commercial protein as a model system, the protocols invite replication and comparison with our results. A commentary discusses principles for modifications in the protocols that may be necessary to optimize application of sedimentation velocity analysis to other self‐associating proteins. ©2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of external calibration factors Basic Protocol 2: Sedimentation velocity experiment for protein self‐association Basic Protocol 3: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

“…Sedimentation velocity analytical ultracentrifugation (SV-AUC) experiments were carried out in a ProteomeLab instrument (Beckman Coulter, Indianapolis) equipped with Rayleigh interference optics, following standard protocols 72 . Samples were inserted in centerpieces with 3 or 12 mm optical pathlength, respectively, with H 2 O as an optical reference, and temperature equilibrated to 20 °C in the rotor at rest, prior to acceleration to 50,000 rpm or 55,000 rpm.…”

Label-free methods for opticalin vitrocharacterization of protein–protein interactions

“…In 2020, the authors’ work resulted in the publication of several protocols for quantitative analysis of protein self-association. 90 …”

“…In 2020, the authors' work resulted in the publication of several protocols for quantitative analysis of protein self-association. 90 At the other extreme of the concentration range, recent developments in fluorescence detected sedimentation velocity AUC enabled measurements at low pM concentrations and of pM K d s, of e.g. antibody-antigen interactions 85 and homodimerization of the glutamate receptor GluA2 amino terminal domain.…”

Determination of specific and non-specific protein–protein interactions for beta-lactoglobulin by analytical ultracentrifugation and membrane osmometry experiments

“…SV experiments were conducted using centerpieces with an optical path length of 12 mm or 3 mm in order to adjust the optical signal. When converting intensity data to absorbance data, pseudo-absorption of each sample was calculated and analyzed as described by Kar et al 82 Following the protocol by Schuck et al, 14,81 SV data was first analyzed with the continuous c(s) model which is implemented in the SEDFIT software (Version 16-1c). 36 The sedimentation coefficient distributions and the weight-averaged sedimentation coefficients for varying protein concentrations were determined using the partial specific volume of 0.751 mL g À1 for BLG as calculated from Sednterp.…”

“…Determination of the dimer dissociation constant was subsequently conducted via fitting the isotherms, i.e. weightaveraged sedimentation coefficient for varying protein concentration at a fixed pH value and salt concentration, using the software SEDPHAT and following the protocol of Zhao et al 14,81…”