Analysis of heterologous interacting systems by sedimentation velocity: curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants.
Analytical ultracentrifugation (AUC) has played and will continue to play an important role in the investigation of protein-protein, protein-DNA and protein-ligand interactions. A major advantage of AUC over other methods is that it allows the analysis of systems free in solution in nearly any buffer without worry about spurious interactions with a supporting matrix. Large amounts of high-quality data can be acquired in relatively short times. Advances in software for the treatment of AUC data over the last decade have eliminated many of the tedious aspects of AUC data analysis, allowing relatively rapid analysis of complicated systems that were previously unapproachable. A software package called sedanal is described that can perform global fits to AUC sedimentation velocity data obtained for both interacting and non-interacting, macromolecular multi-species, multi-component systems, by combining data from multiple runs over a range of sample concentrations and component ratios. Interaction parameters include both forward and reverse rate constants, or equilibrium constants, for each reaction, as well as concentration dependence of both sedimentation and diffusion coefficients. sedanal fits to time-difference data to eliminate time-independent systematic errors inherent in AUC data. The sedanal software package is based on the use of finite-element numerical solutions of the Lamm equation.
Stafford
Walter F
WF
Analytical Ultracentrifugation Research Laboratory, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA. stafford@bbri.org
Sherwood
Peter J
PJ
D000465:Algorithms; D004247:DNA / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008956:Models, Chemical; D009010:Monte Carlo Method; D011506:Proteins / Q000737:chemistry; D012984:Software; D012996:Solutions; D014461:Ultracentrifugation / Q000379:methods*
D008024:Ligands; D046911:Macromolecular Substances; D011506:Proteins; D012996:Solutions; D004247:DNA