How Do Group Ii Chaperonins Distinguish their Partially Folded Substrates from the Native States?

Abstract

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The Archaeal group II chaperonin Mm-Cpn assists the in vitro refolding of the well-characterized β-sheet lens protein human γD-Crystallin (HγD-Crys). The buried cores of this Greek key conformation as well as the domain interface includes a variety of side chains which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, and thus absent from the native protein surface, might be serving as recognition signals. The features tested were a) paired aromatic side chains; b) side chains in the interface between the duplicated domains of HγD-Crys, and c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of HγD-Crys mutants of these residues, during refolding after dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutant proteins indicating that the buried residues were not recognition signals. In fact Mm-Cpn refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the destabilized mutant proteins. The results suggest that Mm-Cpn does not recognize paired aromatic residues, exposed domain interface, or destabilized core - but rather recognizes other features of the partially folded β-sheet, such as an exposed backbone, that are absent or inaccessible in the native state off HγD-Crys. Supported by NIH EY15834 and EY016525.