PLURALITY OF PROTEIN CONFORMATIONS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE MONOMERS PROBED BY HIGH-PRESSURE ELECTROPHORESIS

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 te mperatures below -5-degrees-C or pressures above 1.5 kbar, only a diff use band with low electrophoretic mobility was observed, which is assi gned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0-degrees-C, both the wild type and a mutant in which a positive ly charged Lys at the dimer interface is replaced by a negatively char ged glutamic acid displayed several discrete bands with retardation co efficients larger than that of the dimer. Cross-linking due to oxidati on of cysteines was not the reason for the multiplicity of bands, whic h in addition were independent on the length of the electrophoretic ru n 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 adop t various expanded conformations, which, under the experimental condit ions, are stabilized to the extent necessary to be considered as disti nct chemical species. Gel filtration high performance liquid chromatog raphy analysis of bands eluted from nonstained gels run at 1 kbar (15- degrees-C) and 1.5 kbar (-5-degrees-C), respectively, was performed. I n 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 obt ained at 15-degrees-C, are able to reconstitute a single active confor mation on reassociation.