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Conformational changes occurring in N‐ ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions
Author(s) -
Grand Roger J.A.,
Grant Michael L.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80976-7
Subject(s) - proteolysis , chemistry , divalent , nucleotide , guanine , trypsin , conformational change , biochemistry , biophysics , metal , chymotrypsin , crystallography , stereochemistry , enzyme , biology , organic chemistry , gene
Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N‐ ras p21 in response to ligand binding. It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg 2+ and Ca 2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg 2+ GD. Monovalent cations (Na + or K + ) cannot substitute for Mg 2+ or Ca 2+ . Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation. Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.