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Published online before print February 1, 2006, 10.1110/ps.051856406
Protein Science (2006), 15:533-542. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Methionine oxidation of monomeric {lambda} repressor: The denatured state ensemble under nondenaturing conditions

Preeti Chugha, Harvey J. Sage and Terrence G. Oas

Department of Biochemistry, Duke University, Durham, North Carolina 27710, USA

(RECEIVED September 20, 2005; FINAL REVISION November 18, 2005; ACCEPTED November 21, 2005)

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric {lambda} repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of {alpha}-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

Keywords: repressor proteins; denatured state ensemble; methionine oxidation; hydrodynamic radius; heteronuclear NMR; protein structure/folding; NMR spectroscopy; new methods; circular dichroism

Abbreviations: CD, circular dichroism • HSQC, heteronuclear single quantum coherence • Rh, hydrodynamic radius • {lambda}S, monomeric {lambda} repressor (residues 1–85) • ROS, reactive oxygen species

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051856406.

Reprint requests to: Terrence G. Oas, Department of Biochemistry, 436 Nanaline Duke Building, Box 3711, Duke University Medical Center, Durham, NC 27710, USA; e-mail: oas{at}duke.edu; fax: (919) 681-8862.


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