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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).
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