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The folding landscape of an α‐lytic protease variant reveals the role of a conserved β‐hairpin in the development of kinetic stability
Author(s) -
Truhlar Stephanie M. E.,
Agard David A.
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20525
Subject(s) - folding (dsp implementation) , protein folding , energy landscape , downhill folding , turn (biochemistry) , biophysics , phi value analysis , chemistry , contact order , folding funnel , crystallography , biochemistry , biology , electrical engineering , engineering
Most secreted bacterial proteases, including α‐lytic protease (αLP), are synthesized with covalently attached pro regions necessary for their folding. The αLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the αLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although αLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved β‐hairpin in the C‐terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an αLP variant with a more favorable β‐hairpin turn conformation (αLP β‐turn ). Indeed, αLP β‐turn pro region‐catalyzed folding is faster than that for αLP. However, instead of accelerating spontaneous folding, αLP β‐turn actually unfolds more slowly than αLP. Our data support a model where the β‐hairpin is formed early, but its packing with a loop in the N‐terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of αLP β‐turn , to nearly the same degree as the change between αLP and a faster folding homolog. However, αLP β‐turn has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties. Proteins 2005. © 2005 Wiley‐Liss, Inc.