Probing the binding states of GDP to Cdc42 using urea interaction.
The inactive state of the small G protein Cdc42, the Cdc42.GDP.Mg(2+) ternary complex, was investigated using fluorescence, Mn(2+) substituted electron paramagnetic resonance, and (31)P nuclear magnetic resonance spectroscopy at various urea concentrations. The urea interaction with the protein was used to probe the binding state of GDP.Mg(2+) to Cdc42. Two binding states of the Cdc42.GDP.Mg(2+) ternary complex with different binding stability were observed. The two binding states were characterized by two sets of (31)P resonance of GDP phosphate groups, namely P(alpha) and P(beta), P('alpha), and P('beta). The high populated binding state I (P(alpha) and P(beta)) was more stable and less sensitive to the urea interaction. Yet the population of binding state II (P('alpha) and P('beta)) was lower, and the binding of GDP.Mg(2+) to Cdc42 in this state was more sensitive to the urea interaction. The release of GDP.Mg(2+) from the ternary complex in binding state II was faster than in state I.
Zhao
Ju
J
National Laboratory of Biomacromolecules, Academia Sinica, Beijing 100101, People's Republic of China.
Cheng
Yuan
Y
Wang
Zhixin
Z
Wang
Jinfeng
J
D004578:Electron Spin Resonance Spectroscopy; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D014867:Water; D006153:Guanosine Diphosphate; D008345:Manganese; D014364:Tryptophan; D014508:Urea; D020764:cdc42 GTP-Binding Protein; D008274:Magnesium