An immunological approach to the conformational equilibrium of staphylococcal nuclease

https://doi.org/10.1016/0022-2836(75)90305-8Get rights and content

Abstract

The conformational equilibrium constant, Kconf, of staphylococcal nuclease, describing the equilibrium between the native conformation and non-native or disordered conformations, has been estimated using an immunologic method and an interpretive model. Using goat antisera prepared toward a conformationally disordered nuclease fragment (99–149), antibodies specific for the disordered form of the helix-rich sequence 99 to 126, anti-(99–126)R, were isolated by sequential immunoabsorption. Anti-(99–126)R forms soluble 7 S complexes with fragment (99–149), but this interaction may be inhibited by a large excess of nuclease. By using fragment (99–149) preferentially carbamylated at the α-amino terminus with KN14CO and rabbit anti-goat immunoglobulin to distinguish between antibody-bound and free fragment (99–149), an assay for the quantitation of the degree of inhibition of anti-(99–126)R. (99–149) complex formation by nuclease was developed.

Using a formal analysis based on the hypothesis that nuclease is in a conformational equilibrium between a folded and unfolded form and that anti-(99–126)R binds effectively only to the unfolded form, the Kconf of nuclease was estimated to be 2900. In the presence of the ligands Ca(II), or Ca(II) and thymidine-3′,5′-diphosphate, Kconf values of 6500 and 30,000 to 50,000 were estimated, respectively. The Kconf of nuclease at 4 °C and 39 °C was 3900 and 400, respectively.

References (24)

  • C.B. Anfinsen et al.
  • A. Arnone et al.

    J. Biol. Chem

    (1971)
  • G.A. Bray

    Anal. Biochem

    (1960)
  • B. Furie et al.

    J. Biol. Chem

    (1973)
  • A. Light et al.

    J. Biol. Chem

    (1974)
  • J. Monod et al.

    J. Mol. Biol

    (1965)
  • L. Morávek et al.

    J. Biol. Chem

    (1969)
  • C. Tanford

    Advan. Protein Chem

    (1970)
  • H. Taniuchi et al.

    J. Biol. Chem

    (1969)
  • C.B. Anfinsen

    Science

    (1973)
  • C.B. Anfinsen et al.

    Advan. Protein Chem

    (1975)
  • C.B. Anfinsen et al.
  • Cited by (72)

    • Antigens

      2022, Encyclopedia of Infection and Immunity
    • Folding and association of proteins

      1987, Progress in Biophysics and Molecular Biology
    View all citing articles on Scopus

    Present address: Department of Medicine, Upstate Medical Center, State University of New York, Syracuse, N.Y. 13210, U.S.A.

    View full text