Showing papers by "Emil F. Pai published in 1992"

Simulation of the solution structure of the H-ras p21-GTP complex

Abstract: An unconstrained simulation of the GTP-bound form of the H-ras protein p21 is performed in an aqueous environment with charge-neutralizing counterions. The simulation is compared to the 1.35-A structure of Pai et al. [(1990) EMBO J. 9, 2351] and a proposed alternate structure, in which the loop at residues 60-65 is modeled into a form which may activate a water molecule for the GTP hydrolysis. The simulation suggests that some protein intermolecular H-bond contacts which are present in the crystal structure are lost in the solvation process and this loss may lead to localized refolding of the molecule. For instance, we find that the gamma-phosphate of the GTP has somewhat weaker contact with the protein in the simulation structure. The antiparallel beta-sheet (residues 38-57) partially melts. The 60-65 loop, which is hypervariable in the X-ray study, is initially relatively distant from the gamma-phosphate region. However, this loop moves so as to sample the space around the gamma-phosphate. For a significant fraction of the simulation time, forms similar to the alternate structure are observed, and a water molecule is localized near the hydrolytic site. The molecular dynamics simulations of p21-GTP in solution support a postulated hydrolysis mechanism for the biological inactivation of the nucleotide complex based on crystallographic data.

Studies on the structure and mechanism of H-ras p21.

Abstract: Current knowledge of the structure of H-ras p21 is reviewed with particular emphasis on the interaction between guanine nucleotides and the active site of the protein. The nature of the conformational change induced by GTP hydrolysis is discussed. The major change is seen in the region known as the effector loop (loop 2), with significant but less well-defined changes occurring in loop 4, which is implicated in the GTPase reaction. Other evidence concerning the mechanism of GTP hydrolysis and its activation by GAP (GTPase-activating protein) is also discussed. Evidence regarding the rate limiting step in the p21 GTPase reaction, and the manner in which this and possibly other steps are accelerated by GAP, is inconclusive.

Time-resolved crystallography on H-ras p21

Abstract: We describe here the results obtained to date on a project aimed at characterizing the changes occurring in the protein product (p21) of the H-ras proto-oncogene during and as a result of hydrolysis of GTP at its active site. The approach used involves crystallization of p21 with a photosensitive precursor of GTP (caged GTP) at the active site followed by generation of GTP by photolysis and collection of X-ray diffraction data using the Laue method at a synchrotron source. The structure of p21 complexed with a single diastereomer of caged GTP is presented here. In contrast to crystals obtained with mixed diastereomers, the nucleotide appears to bind in a manner which is very similar to that of other guanine nucleotides (GDP, GTP, GppNHp). The current state of time resolved structural experiments using these crystals is presented.