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Protein Science (2004), 13:830-841. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Zhengrong W. Yang1, Susan W. Tendian1, W. Michael Carson1,2, Wayne J. Brouillette1,3, Lawrence J. Delucas1,4 and Christie G. Brouillette1,5

1 Center for Biophysical Sciences and Engineering,
2 Department of Biomedical Engineering, School of Engineering,
3 Department of Chemistry, School of Natural Sciences and Mathematics,
4 Department of Optometry,
5 Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham, Birmingham, Alabama 35294-4400, USA

(RECEIVED July 31, 2003; FINAL REVISION October 31, 2003; ACCEPTED October 31, 2003)



Abstract

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD+ synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme’s catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, Ka, equal to 5.6 x 106/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer {leftrightarrows} folded monomer {leftrightarrows} unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large Ka down to ~106/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.

Keywords: differential scanning calorimetry; analytical ultracentrifugation; compound screening; dimerization; structural cooperativity; protein folding; DMSO

Reprint requests to: Christie G. Brouillette, Center for Biophysical Sciences and Engineering, 1530 3rd Avenue South, CBSE 234, Birmingham, AL 35294-4400, USA; e-mail: Christie{at}uab.edu; fax: (205) 934-3352.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03330104.

6 That is, for a process F {leftrightarrows} U -> I, where I is the irreversibly changed unfolded protein, if the rate of formation of I is slower than the rate of reformation of F from U, then equilibrium between F and U will be achieved during the transition of the first DSC scan even though a repeat DSC scan will show no transition.

7 Possible changes in protonation, or the release of buried interfacial water, upon dimer dissociation cannot be taken into account when calculating the {Delta}Cp based on changes in solvent accessible surface area, although they would affect the {Delta}Cp, if present.

8 The excess heat capacity curve obtained by DSC can be mathematically fit with or without the progressive baseline shift that occurs as a consequence of the change in heat capacity, {Delta}Cpu, between the folded and unfolded states. To simplify the DSC curve fitting, the progressive baseline was subtracted, which sets {Delta}Cpu to 0, and floors the pre- and post-transition baselines to 0.


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A. Tjernberg, N. Markova, W. J. Griffiths, and D. Hallen
DMSO-Related Effects in Protein Characterization
J Biomol Screen, March 1, 2006; 11(2): 131 - 137.
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