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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).
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