Protein Folding in vitro
Alan Fersht, Centre
for Protein Engineering, University of Cambridge
The game plan of the experimentalist to solve any mechanism is
staightforward: to determine the structures of all of the
and transition states, including starting materials and products,
the reaction pathway. Whereas this can be a simple problem in
classical chemistry where just a few bonds change in a reaction,
is an extremely difficult problem in protein folding because the
of the structure of the protein changes during the reaction and
usually extremely difficult to isolate stable intermediates.
Nevertheless, this has been accomplished to a high degree for a
small proteins using a combination of NMR, protein engineering
kinetics. The structure of the ensemble that constitutes the
denatured state may be probed by NMR. The structures of
states and unstable intermediates may be analysed by the
"protein engineering method" to give structures at
resolution. A series of such experiments on the proteins barnase,
chymotrypsin inhibitor 2 and barstar, have identified the
of a nucleation mechanism, "nucleation-condensation" in
folding. The background, methods and results will be discussed in
Protein folding and stability: the pathway of folding of barnase.
Sixth Datta Lecture. A. R. Fersht, FEBS Letters 325, 5-16 (1993).
The structure of the transition state for the folding/unfolding
barley chymotrypsin inhibitor 2 and its implications for
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
Characterizing Transition States in Protein Folding: An Essential
in the Puzzle. A. R. Fersht, Current Opinion in Structural
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
A. R. Fersht Proc. Natl. Acad. Sci. U.S.A. 92, 10668-10672
The Folding Pathway of a Protein at High Resolution from
to Seconds. B. Nölting, R. Golbik, J.-L. Neira, A. S.
G. Schreiber and A. R. Fersht Proc. Natl. Acad. USA 94, 826-830
The structure of the transition state for folding of chymotrypsin
inhibitor 2 analysed by protein engineering methods: Evidence for
nucleation-condensation mechanism for protein folding. L. S.
D. E. Otzen and A. R. Fersht J. Mol. Biol. 254, 260-288 (1995).
A comparison of the pH-, urea, and temperature-denatured states
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.
K.-B. Wong and A. R. Fersht Proc. Natl. Acad. USA 93, 10600-10603