Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s).
We describe algorithms for solving the Lamm equations for the reaction-diffusion-sedimentation process in analytical ultracentrifugation, and examine the potential and limitations for fitting experimental data. The theoretical limiting case of a small, uniformly distributed ligand rapidly reacting with a larger protein in a "constant bath" of the ligand is recapitulated, which predicts the reaction boundary to sediment with a single sedimentation and diffusion coefficient. As a consequence, it is possible to express the sedimentation profiles of reacting systems as c(s) distribution of noninteracting Lamm equation solutions, deconvoluting the effects of diffusion. For rapid reactions, the results are quantitatively consistent with the "constant bath" approximation, showing c(s) peaks at concentration-dependent positions. For slower reactions, the deconvolution of diffusion is still partially successful, with c(s) resolving peaks that reflect the populations of sedimenting species. The transition between c(s) peaks describing reaction boundaries of moderately strong interactions (K(D) approximately 10(-6) M) or resolving sedimenting species was found to occur in a narrow range of dissociation rate constant between 10(-3) and 10(-4) s(-1). The integration of the c(s) peaks can lead to isotherms of species populations or s-value of the reaction boundary, respectively, which can be used for the determination of the equilibrium binding constant.
Dam
Julie
J
Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, Maryland, USA.
Velikovsky
Carlos A
CA
Mariuzza
Roy A
RA
Urbanke
Claus
C
Schuck
Peter
P
D000465:Algorithms; D001703:Biophysics / Q000379:methods*; D004058:Diffusion; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances / Q000737:chemistry; D015233:Models, Statistical; D008968:Molecular Conformation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D013053:Spectrophotometry; D013997:Time Factors; D014461:Ultracentrifugation
D008024:Ligands; D046911:Macromolecular Substances; D011506:Proteins