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Study of the effect of Ca(OAc)2 concentration on the state of the Allosteric Switch in Ras GTPase
Author(s) -
Johnson Christian William,
Davis Kathleen,
Mattos Carla
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.831.17
Subject(s) - allosteric regulation , gtpase , gtp' , chemistry , allosteric enzyme , small gtpase , salt bridge , signal transduction , mutant , g protein , biophysics , microbiology and biotechnology , biochemistry , enzyme , biology , gene
Ras GTPase cycles between GDP inactive and GTP active forms and is of great importance in signal transduction that regulates cell proliferation, differentiation and survival. Although signaling is largely attenuated by the presence of GTPase activating proteins (GAPs), our group has recently discovered and allosteric mechanism that may promote hydrolysis of GTP in the presence of Raf and possibly other tight binding effectors, demonstrating subtle levels of regulation of the GTP‐bound protein. The allosteric switch is ON, with complete ordering of the active site, in crystals soaked in 100mM Ca(OAc) 2 , which binds to an allosteric site remote from the catalytic center. In the presence of CaCl 2 the allosteric switch is OFF. Ca(OAc) 2 binding is mediated by arginine 97 (R97) through salt bridge interactions with acetate. Here we show that R97A, which cannot bind acetate, is in the OFF state in the presence of 245 mM Ca(OAc) 2 but switches to the ON state at 365 mM Ca(OAc) 2 . The R97F mutant is observed in the OFF state in the presence of 200mM Ca(OAc) 2 . It is clear that the bulk solvent composition has an important effect in the state of the allosteric switch in R97A. The soaking of crystals in 365 mM Ca(OAc) 2 will test whether this is also the case for R97F and other allosteric site mutants.