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J. Biol. Chem., Vol. 283, Issue 40, 27121-27129, October 3, 2008

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Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis^*

Raul Perez-Jimenez¹, Arun P. Wiita, David Rodriguez-Larrea^¶, Pallav Kosuri^||, Jose A. Gavira^**, Jose M. Sanchez-Ruiz^¶, and Julio M. Fernandez²

From the Department of Biological Sciences and the Graduate Program in Neurobiology and Behavior, Columbia University, New York, New York 10027, the ^¶Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, 18071, Granada, Spain, the ^||Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10027, and the ^**Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Cientificas-Universidad de Granada), Parque Tecnologico Ciencias de la Salud, 18100 Granada, Spain

Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.

Received for publication, May 15, 2008 , and in revised form, August 6, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL66030 and HL61228 (to J. M. F.). This work was also supported by Grant BIO2006-07332 from the Spanish Ministry of Education and Science, FEDER Funds, and Grant CVI-771 from Junta de Andalucia (to J. M. S.-R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ Postdoctoral Fellow of the Spanish Ministry of Education and Science.

² To whom correspondence should be addressed: Dept. of Biological Sciences, Columbia University, 1011A Fairchild Center, 1212 Amsterdam Ave., MC 2449, New York, NY 10027. Tel.: 212-854-9474; Fax: 212-854-9606; E-mail: jfernandez{at}columbia.edu.

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