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Published online before print January 10, 2004, 10.1110/ps.03336804
Protein Science (2004), 13:381-390. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability

Stephanie M.E. Truhlar1, Erin L. Cunningham2 and David A. Agard1,2,3

1 Graduate Program in Chemistry and Chemical Biology,
2 Graduate Group in Biophysics, and
3 Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, 94143-2240, USA

(RECEIVED July 28, 2003; FINAL REVISION October 15, 2003; ACCEPTED October 16, 2003)



Abstract

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and {alpha}-lytic protease ({alpha}LP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of {alpha}LP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and {alpha}LP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and {alpha}LP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4–8 in protease resistance is accompanied by a cost of ~105 in the spontaneous folding rate and ~5–9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.

Keywords: Streptomyces griseus protease B; protein folding; pro region; kinetic stability; protein evolution

Abbreviations: SGPB, Streptomyces griseus protease B • {alpha}LP, {alpha}-lytic protease • Pro{alpha}LP, the {alpha}-lytic protease pro region • ProSGPB, the S. griseus protease B pro region • Nat{alpha}LP, the native state of {alpha}-lytic protease • NatSGPB, the native state of S. griseus protease B • IntSGPB, the intermediate state of S. griseus protease B • CD, circular dichroism • GndHCl, guanidine hydrochloride • TLCK, tosyl-L-lysine chloromethyl ketone

Reprint requests to: David A. Agard, Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, Room S412, San Francisco, CA 94143-2240, USA; e-mail: agard{at}msg.ucsf.edu; fax: (415) 476-1902.

Supplemental material: See www.proteinscience.org

Article published online ahead of print.

Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03336804.


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