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PEDS Advance Access published online on March 20, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl007
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received February 16, 2006
Accepted February 17, 2006

Short Communication

Generation and analysis of proline mutants in protein G

Eun Jung Choi 1 and Stephen L. Mayo 2 *

1 Division of Biology, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA, USA
2 Division of Biology, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA, USA; Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125, USA

* To whom correspondence should be addressed.
Stephen L. Mayo, E-mail: steve{at}mayo.caltech.edu


  Abstract

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.

Keywords: proline; protein; design; protein G; protein stability.
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