title: The use of analytical sedimentation velocity to extract thermodynamic linkage.
abstract: For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Cole
authors.forename: John J
authors.initials: JJ
authors.lastname: Correia
authors.forename: Walter F
authors.initials: WF
authors.lastname: Stafford
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000379:methods*; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011506:Proteins; D000069816:SEC Translocation Channels; D019892:eIF-2 Kinase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins