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Mechanism of Protein Folding in vivo Alan Fersht, Centre for Protein Engineering, University of Cambridge Protein folding in vivo is aided by molecular chaperones. These are proteins that bind to denatured states of proteins and prevent them misfolding and aggregating. The most interesting protein regarding folding is GroEL which is a typical member of the Hsp60 or Cpn60 class of molecular chaperonins. It consists of 14 x 58kDa subunits arranged in two-stacked seven-member rings which have a large central cavity. In vivo a co-chaperonin GroES is also required as is the hydrolysis of ATP. The mechanism of GroEL is controversial. One school proposes that the central cavity acts as a folding cage in which a single molecule of denatured protein can fold in isolation. The opposing school postulates that GroEL is an active unfoldase that catalyses the unfolding of misfolding and aggregated states. A series of experiments examining the folding of barnase and CI2 in the presence of GroEL showed that both proteins can fold from their denatured states when bound to chaperone. The chaperonin actually slows down folding by binding to the denatured state but it increases the yield of active protein. By measuring all of the rate constants under a variety of conditions a complex mechanism, involving energy transduction and allosteric changes, may be derived. GroEL in vivo appeared to act as a folding and annealing cage. But, a specific fragment of GroEL, consisting of about 150 residues, has been produced that efficiently chaperones the refolding of typical substrates of GroEL, without a requirement for the hydrolysis of ATP. Further, the fragment is very efficient when immobilised on a solid support. This finding has two consequences. First, in combination with data on hydrogen-deuterium exchange, it shows that the principal activity of GroEL is to act as an unfoldase. Second, the immobilised GroEL is of biotechnological importance because it provides a reusable reagent for the renaturation of proteins. The fragment may be crystallised and its structure solved at high resolution. A fusion polypeptide from one molecule in the crystal binds in the active site of another to provide a detailed structure of the enzyme-substrate complex, which provides a structural basis for the unfolding activity. The folding of GroEL-bound barnase as a model for chaperonin-mediated protein folding. F. J. Corrales and A. R. Fersht Proc. Natl. Acad. Sci. U.S.A. 92, 5326-5330 (1995). Towards a mechanism for GroELˇGroES chaperone activity: an "ATPase-gated and -pulsed folding and annealing cage". F. J. Corrales and A. R. Fersht Proc. Natl. Acad. Sci. U.S.A. 93, 4509-13 (1996). Catalysis of Amide Protein Exchange by Molecular Chaperones GroEL and SecB. R. Zahn, S. Perrett, G. Stenberg and A. R. Fersht, Science 271, 642-645 (1996). Chaperone Activity and Structure of Monomeric Polypeptide Binding Domains of GroEL. R. Zahn, A. M. Buckle, S. Perrett, C. M. Johnson, F. J. Corrales, R. Golbik and A. R. Fersht Proc. Natl. Acad. USA 93, 15024-15029 (1996). The Folding Pathway of a Protein at High Resolution from Microseconds to Seconds. B. Nölting, R. Golbik, J.-L. Neira, A. S. Soler-Gonzalez, G. Schreiber and A. R. Fersht Proc. Natl. Acad. USA 94, 826-830 (1997). Chaperone Activity and Structure of Monomeric Polypeptide Binding Domains of GroEL. R. Zahn, A. M. Buckle, S. Perrett, C. M. Johnson, F. J. Corrales, R. Golbik and A. R. Fersht Proc. Natl. Acad. USA 93, 15024-15029 (1996). Refolding chromatography with immobilized mini-chaperones. Myriam M. Altamirano, Ralph Golbik, Ralph Zahn, Ashley M. Buckle and Alan R. Fersht Proc. Natl. Acad. USA, in press (1997). A Structural Model for GroEL-Polypeptide Recognition. A. M. Buckle, R. Zahn and A. R. Fersht Proc. Natl. Acad. USA, in press (1997). ## Back to the list of abstracts