Nonequilibrium self-association of a cpn60 chaperonin induced by tryptophan mutation

Abstract

The tryptophan mutant Y203W of the bacterial GroEL (cpn60) was studied with regard to its hydrodynamic integrity and its oligomeric state. Sedimentation equilibrium using MSTARI gave a weight-average molecular weight of (905,000 ± 33,000) Da. This is in excellent agreement with results from sedimentation velocity, which revealed three distinct species (19.6S, 26.5S and 38S) in the same proportions by weight for six different loading concentrations, corresponding to 14- and 28-mer subunit compositions with a smaller dissociation product. The relative amounts of each species present, (23.0 ± 0.8)%, (61.5 ± 2.9)% and (15.5 ± 3.0)%, yielded an estimated weight-average molecular weight of about 870,000 Da. From this we conclude that the tryptophan mutation at the Y203 location causes significant irreversible self-association under the conditions used here, and appears to be yet another example of how sedimentation analysis can be used to probe the effects of a single amino acid substitution in a protein on the conformation and hence the oligomeric state of a protein assembly.