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R-Lytic Protease Precursor: Characterization of a Structured Folding Intermediate D. Eric Anderson,, Reuben J. Peters,,| Barry Wilk, and David A. Agard*
 

Summary: R-Lytic Protease Precursor: Characterization of a Structured Folding Intermediate
D. Eric Anderson,, Reuben J. Peters,,| Barry Wilk, and David A. Agard*
The Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, UniVersity of California,
San Francisco, California 94143-0448
ReceiVed September 10, 1998; ReVised Manuscript ReceiVed January 11, 1999
ABSTRACT: The bacterial R-lytic protease (RLP) is synthesized as a precursor containing a large N-terminal
pro region (Pro) transiently required for correct folding of the protease [Silen, J. L., and Agard, D. A.
(1989) Nature 341, 462-464]. Upon folding, the precursor is autocatalyticly cleaved to yield a tight-
binding inhibitory complex of the pro region and the fully folded protease (Pro/RLP). An in vitro purification
and refolding protocol has been developed for production of the disulfide-bonded precursor. A combination
of spectroscopic approaches have been used to compare the structure and stability of the precursor with
either the Pro/RLP complex or isolated Pro. The precursor and complex have significant similarities in
secondary structure but some differences in tertiary structure, as well as a dramatic difference in stability.
Correlations with isolated Pro suggest that the pro region part of the precursor is fully folded and acts to
stabilize and structure the RLP region. Precursor folding is shown to be biphasic with the fast phase
matching the rate of pro region folding. Further, the rate-limiting step in oxidative folding is formation
of the disulfide bonds and autocatalytic processing occurs rapidly thereafter. These studies suggests a
model in which the pro region folds first and catalyzes folding of the protease domain, forming the active
site and finally causing autocatalytic cleavage of the bond separating pro region and protease. This last
processing step is critical as it allows the protease N-terminus to rearrange, providing the majority of net

  

Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco

 

Collections: Biotechnology; Biology and Medicine