Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase.

Academic Article

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, K(a), equal to 5.6 x 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 approximately 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.
  • Published In

  • Protein Science  Journal
  • Keywords

  • Algorithms, Amide Synthases, Bacillus subtilis, Bacterial Proteins, Calorimetry, Differential Scanning, Dimethyl Sulfoxide, Enzyme Stability, Models, Chemical, Models, Molecular, Protein Conformation, Protein Denaturation, Protein Folding, Protein Renaturation, Protein Structure, Quaternary, Protein Subunits, Recombinant Proteins, Thermodynamics, Ultracentrifugation
  • Digital Object Identifier (doi)

    Authorlist

  • Yang ZW; Tendian SW; Carson WM; Brouillette WJ; Delucas LJ; Brouillette CG
  • Start Page

  • 830
  • End Page

  • 841
  • Volume

  • 13
  • Issue

  • 3