-Lytic Protease Precursor: Characterization of a Structured Folding Intermediate
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- 作者：D. Eric Anderson ; Reuben J. Peters ; Barry Wilk ; and David A. Agard
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：April 13, 1999
- 年：1999
- 卷：38
- 期：15
- 页码：4728 - 4735
- 全文大小：101K
- 年卷期：v.38,no.15(April 13, 1999)
- ISSN：1520-4995
文摘
The bacterial 
-lytic protease (LP) is synthesized as a precursor containing a large N-terminalpro 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/LP). An in vitro purificationand refolding protocol has been developed for production of the disulfide-bonded precursor. A combinationof spectroscopic approaches have been used to compare the structure and stability of the precursor witheither the Pro/LP complex or isolated Pro. The precursor and complex have significant similarities insecondary 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 tostabilize and structure the LP region. Precursor folding is shown to be biphasic with the fast phasematching the rate of pro region folding. Further, the rate-limiting step in oxidative folding is formationof the disulfide bonds and autocatalytic processing occurs rapidly thereafter. These studies suggests amodel in which the pro region folds first and catalyzes folding of the protease domain, forming the activesite and finally causing autocatalytic cleavage of the bond separating pro region and protease. This lastprocessing step is critical as it allows the protease N-terminus to rearrange, providing the majority of netstabilization of the product Pro/LP complex.
-lytic protease (LP) is synthesized as a precursor containing a large N-terminalpro 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/LP). An in vitro purificationand refolding protocol has been developed for production of the disulfide-bonded precursor. A combinationof spectroscopic approaches have been used to compare the structure and stability of the precursor witheither the Pro/LP complex or isolated Pro. The precursor and complex have significant similarities insecondary 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 tostabilize and structure the LP region. Precursor folding is shown to be biphasic with the fast phasematching the rate of pro region folding. Further, the rate-limiting step in oxidative folding is formationof the disulfide bonds and autocatalytic processing occurs rapidly thereafter. These studies suggests amodel in which the pro region folds first and catalyzes folding of the protease domain, forming the activesite and finally causing autocatalytic cleavage of the bond separating pro region and protease. This lastprocessing step is critical as it allows the protease N-terminus to rearrange, providing the majority of netstabilization of the product Pro/LP complex.