title: Crowding by trisaccharides and the 2:1 cytochrome c-cytochrome c peroxidase complex.
abstract: Proteins are designed to function under crowded conditions where the solute concentration can reach 400 g/L, but they are almost always studied in dilute solutions. To address this discrepancy, we have undertaken a series of studies to determine the effects of high solute concentrations on the thermodynamics of protein equilibria. Recently, we used isothermal titration calorimetry (ITC) to show that high concentrations of mono-, di-, and tetrasaccharides have a small stabilizing effect on the crystallographically defined cytochrome c binding site on yeast ferricytochrome c peroxidase [Morar, A. S., Wang, X., and Pielak, G. J. (2001) Biochemistry 40, 281-285]. Here, we use this technique to show that trisaccharides increase the apparent thermodynamic binding constants for both cytochrome c binding sites on the peroxidase. Mutagenesis studies confirm that the second site includes Asp 148 on the peroxidase. Binding of both cytochrome c molecules is exothermic. The data are interpreted by assuming either the presence or absence of intersite interactions.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
authors.forename: Artemiza S
authors.initials: AS
authors.lastname: Morar
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Pielak
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002151:Calorimetry; D003574:Cytochrome c Group / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013816:Thermodynamics; D014312:Trisaccharides / Q000737:chemistry*
chemical_list: D003574:Cytochrome c Group; D014312:Trisaccharides; D001224:Aspartic Acid; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase