title: Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution.
abstract: We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse (15)N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide (15)N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.
authors.affiliation: Departament de Química Orgànica, Universitat de Barcelona, Martí i Franquès, 1-11, 08028 Barcelona, Spain.
authors.forename: Pau
authors.initials: P
authors.lastname: Bernadó
authors.forename: Tomas
authors.initials: T
authors.lastname: Akerud
authors.forename: José
authors.initials: J
authors.lastname: García de la Torre
authors.forename: Mikael
authors.initials: M
authors.lastname: Akke
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
mesh_terms: D000818:Animals; D002417:Cattle; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions
chemical_list: D012996:Solutions; D017027:Protein Tyrosine Phosphatases