title: Plurality of protein conformations of ribulose-1,5-bisphosphate carboxylase/oxygenase monomers probed by high pressure electrophoresis.
abstract: We used hydrostatic pressure in the range of 1 to 2 kbar, coupled with polyacrylamide gel electrophoresis, to investigate the properties of monomers of dimeric ribulose bisphosphate carboxylase/oxygenase. At temperatures below -5 degrees C or pressures above 1.5 kbar, only a diffuse band with low electrophoretic mobility was observed, which is assigned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0 degree C, both the wild type and a mutant in which a positively charged Lys at the dimer interface is replaced by a negatively charged glutamic acid displayed several discrete bands with retardation coefficients larger than that of the dimer. Cross-linking due to oxidation of cysteines was not the reason for the multiplicity of bands, which in addition were independent on the length of the electrophoretic run in the range of 1-3 h. Binding of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid to the pressure dissociated monomers stabilized the unfolded conformations. We propose that the dissociated monomers adopt various expanded conformations, which, under the experimental conditions, are stabilized to the extent necessary to be considered as distinct chemical species. Gel filtration high performance liquid chromatography analysis of bands eluted from nonstained gels run at 1 kbar (15 degrees C) and 1.5 kbar (-5 degrees C), respectively, was performed. In both cases the dimeric structure was fully recovered, along with the spectroscopic properties and catalytic activity characteristic of the native dimer, indicating that the unresolved unfolded conformers that appear at -5 degrees C, as well as the set of discrete conformers obtained at 15 degrees C, are able to reconstitute a single active conformation on reassociation.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana 61801.
authors.forename: L
authors.initials: L
authors.lastname: Erijman
authors.forename: A A
authors.initials: AA
authors.lastname: Paladini
authors.forename: G H
authors.initials: GH
authors.lastname: Lorimer
authors.forename: G
authors.initials: G
authors.lastname: Weber
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005971:Glutamates; D018698:Glutamic Acid; D006874:Hydrostatic Pressure; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D005971:Glutamates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine