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Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD + synthetase
Author(s) -
Yang Zhengrong W.,
Tendian Susan W.,
Carson W. Michael,
Brouillette Wayne J.,
Delucas Lawrence J.,
Brouillette Christie G.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03330104
Subject(s) - dimer , chemistry , cooperativity , monomer , crystallography , nad+ kinase , dimethyl sulfoxide , sedimentation equilibrium , stereochemistry , enzyme , organic chemistry , biochemistry , polymer
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, K a , equal to 5.6 × 10 6 /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 ⇆ folded monomer ⇆ unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K a down to ∼10 6 /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.