Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation

Abstract

This unit introduces the basic principles and practice of sedimentation velocity analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The analytical tools described include sedimentation coefficient and molar mass distributions, multi-signal sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource.

Tubulin Dimer Reversible Dissociation

“…In principle, if dissociation is slow relative to the time of the centrifugation, monomer and dimer species will cleanly separate and appear as single peaks in the c(s) distribution whose s w values do not vary with total concentration. If dissociation is fast relative to the centrifugation, a single species with s value intermediate between that of monomer and dimer will be observed (40,54). An advantage of fluorescence polarization is that it allows direct real time examination of the kinetics of processes under study (55).…”

“…SV experiments were carried out at 50,000 rpm in an 8-hole An-50 Ti rotor at 20°C, using 400 l of samples in 12-mm double-sector charcoal-filled Epon centerpieces, using standard procedures as described in more detail elsewhere (40). To accommodate a large number of samples in absorbance experiments, data were acquired in the intensity mode (41).…”

“…Thus, for a monomer-dimer system, slow dissociation kinetics will produce two peaks centered at the s values of the monomer and dimer species, with changing amplitudes as a function of concentration. In contrast, fast dissociation kinetics will produce a single broad peak reflecting an effective particle with a time-average state with increasing s values and amplitudes, as a function of increasing protein concentration (40). This is the basis for modeling of SV data using Lamm equation solutions coupled to reaction fluxes (LEq), which provides thermodynamic as well as kinetic information for the underlying chemical equilibrium.…”

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

“…The cell assembly in the sedimentation velocity experiments contained identical sample and reference buffer volumes of 300 l, was placed in a rotor, and temperature equilibrated at rest at 20°C for 2 h before it was accelerated from 0 to 50,000 rpm. Rayleigh interference optical data were collected at 1-min intervals for 12 h. The velocity data were modeled with diffusion-deconvoluted sedimentation coefficient distributions c(s) in SEDFIT (20,21), using algebraic noise decomposition and with signal-average frictional ratio and meniscus position refined with non-linear regression. The s-value was corrected for time, temperature, and radial position, and finite acceleration of the rotor was accounted for in the evaluation of Lamm equation solutions (22,23).…”

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

“…The absorbance signal was monitored in a continuous mode with a step size of 0.003 cm and a single reading per step. Sedimentation coefficients were calculated from SV profiles using the program SEDFIT (53). The continuous c(s) distributions were calculated assuming a direct sedimentation boundary model using the Lamm equation with maximum entropy regularization at a confidence level of 1 S.D.…”