Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions.
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 Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GDP. Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+. 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.
Grand
R J
RJ
Department of Cancer Studies, The Medical School, University of Birmingham, B15 2TJ, England.
Grant
M L
ML
D002460:Cell Line; D002918:Chymotrypsin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006150:Guanine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000648:ultrastructure; D016283:Proto-Oncogene Proteins p21(ras); D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D006150:Guanine Nucleotides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin; D014357:Trypsin; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D008274:Magnesium