GROEL-MEDIATED PROTEIN FOLDING

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DESCRIPTION: This proposal, which is a continuation of an earlier funded proposal, is aimed at further defining the role of GroEL and its cochaperonin, GroES, in protein folding in vivo. Unfolded polypeptides form ternary complexes with GroEL/GroEs and bind either trans, i.e., on the opposite side from GroES, or bind cis, i.e., on the same side as GroES within a central chamber formed from GroEL and GroES. Folding may proceed differently at the trans and cis sides. A major goal (Aim 1) is to determine precisely the perecentage and identity of the E. coli proteins which are substrates of GroEL. These studies will exploit rapid onset temperature-sensitive mutants of GroEL to be constructed in the P.I.'s laboratory, to address a variety of questions: Are large proteins (>60 kDa) which should be incapable of binding cis because of their size also substrates of GroEL? if so, do their misfolded forms bind productively in trans, i.e, are they unfolded and released by GroEL for a subsequent round of folding in solution? Are small proteins (<10 kDa), which because of their size potentially can fold more rapidly than they can bind GroEL, also substrates of GroEL/GroES? If so, the P.I. will use such proteins for mechanistic studies by deuterium exchange and NMR spectroscopy to elucidate cis-mediated folding. A second goal (Aim 2) is to observe directly the conformation changes associated with substrate binding to GroEL. Protease and tryptophan fluorescence studies are proposed to characterize conformational changes which accompany the binding of metastable rubisco and mitochondrial malate dehydrogenase (mMDH) forms to GroEL. Stopped-flow pyrene fluorescence studies of pyrene-labeled rubisco and dihydrofolate reductase (DHFR) are also proposed. The latter studies would permit quantitative comparisons of unfolding kinetics (monitored by fluorescence changes) with binding kinetics (monitored by fluorescence anisotropy measurements) and help establish whether "native-like" intermediates need to first unfold in order to bind GroEL or whether they can bind GroEL and then unfold. A third goal (Aim 3) is to determine the fate of substrate molecules starting in a cis ternary complex with GroEL/GroES. Can polypeptides, for example, leave the cis side in a non-native state, or are they released only in the native state with release of non-native forms occuring only from the trans side (as is hypothesized by some current models)? A "cis-only" chaperonin complex constructed in the P.I.'s laboratory, in conjunction with GroEL-mutant "traps" that are capable of binding but not releasing non-native polypeptides, will be used to test these hypotheses.