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Mechanism of Protein Folding in vitro Alan Fersht, Centre for Protein Engineering, University of Cambridge The game plan of the experimentalist to solve any mechanism is quite staightforward: to determine the structures of all of the intermediate and transition states, including starting materials and products, on the reaction pathway. Whereas this can be a simple problem in classical chemistry where just a few bonds change in a reaction, this is an extremely difficult problem in protein folding because the whole of the structure of the protein changes during the reaction and it is usually extremely difficult to isolate stable intermediates. Nevertheless, this has been accomplished to a high degree for a few small proteins using a combination of NMR, protein engineering and kinetics. The structure of the ensemble that constitutes the denatured state may be probed by NMR. The structures of transition states and unstable intermediates may be analysed by the so-called "protein engineering method" to give structures at almost atomic resolution. A series of such experiments on the proteins barnase, chymotrypsin inhibitor 2 and barstar, have identified the importance of a nucleation mechanism, "nucleation-condensation" in protein folding. The background, methods and results will be discussed in this lecture. Protein folding and stability: the pathway of folding of barnase. The Sixth Datta Lecture. A. R. Fersht, FEBS Letters 325, 5-16 (1993). The structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. D. E. Otzen, L. S. Itzhaki, N. F. elMasry, S. E. Jackson and A. R. Fersht Proc. Natl. Acad. Sci. U.S.A. 91 10422-10425 (1994). Characterizing Transition States in Protein Folding: An Essential Step in the Puzzle. A. R. Fersht, Current Opinion in Structural Biology, 5, 79-84 (1995). Nucleation Mechanisms in Protein Folding. A. R. Fersht, Current Opinion in Structural Biology, 7, 3-9 (1997). Submillisecond events in protein folding. B. Nolting, R. Golbik and A. R. Fersht Proc. Natl. Acad. Sci. U.S.A. 92, 10668-10672 (1995). 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). The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: Evidence for a nucleation-condensation mechanism for protein folding. L. S. Itzhaki, D. E. Otzen and A. R. Fersht J. Mol. Biol. 254, 260-288 (1995). A comparison of the pH-, urea, and temperature-denatured states of barnase by heteronuclear NMR: Implications for the initiation of protein folding. V. L. Arcus, S. Vuilleumier, S. M. V. Freund, M. Bycroft and A. R. Fersht J. Mol. Biol. 254, 305-321 (1995). Initiation Sites of Protein Folding by NMR Analysis. S. M. V. Freund, K.-B. Wong and A. R. Fersht Proc. Natl. Acad. USA 93, 10600-10603 (1996). ## Back to the list of abstracts