Remodeling of the Folding Free Energy Landscape of Staphylococcal Nuclease by Cavity-Creating Mutations
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- 作者：Julien Roche ; Mariano Dellarole ; Jose A. Caro ; Ewelina Guca ; Douglas R. Norberto ; Yinshan Yang ; Angel E. Garcia ; Christian Roumestand ; Bertrand Garc铆a-Moreno ; Catherine A. Royer
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：November 27, 2012
- 年：2012
- 卷：51
- 期：47
- 页码：9535-9546
- 全文大小：667K
- 年卷期：v.51,no.47(November 27, 2012)
- ISSN：1520-4995
文摘
The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, 螖+PHS. The stabilizing mutations of 螖+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the 螖+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of 螖+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the 螖+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.