Abstract：
:The bacterial alpha-lytic protease (alphaLP) 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/alphaLP). 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/alphaLP 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 alphaLP 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 stabilization of the product Pro/alphaLP complex.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Anderson DE,Peters RJ,Wilk B,Agard DAdoi
10.1021/bi982165esubject
Has Abstractpub_date
1999-04-13 00:00:00pages
4728-35issue
15eissn
0006-2960issn
1520-4995pii
bi982165ejournal_volume
38pub_type
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