title: Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions.
abstract: Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.
authors.affiliation: Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, Texas.
authors.forename: Laurel
authors.initials: L
authors.lastname: Hoffman
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Physics and Astronomy, Clemson University, Clemson, South Carolina.
authors.forename: Hugo
authors.initials: H
authors.lastname: Sanabria
authors.affiliation: Department of Physics, University of Houston, Houston, Texas; The Center for Theoretical Biological Physics, Rice University, Houston, Texas.
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Cheung
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas.
authors.forename: John A
authors.initials: JA
authors.lastname: Putkey
authors.affiliation: Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, Texas. Electronic address: m.n.waxham@uth.tmc.edu.
authors.forename: M Neal
authors.initials: MN
authors.lastname: Waxham
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011485:Protein Binding
chemical_list: D002147:Calmodulin