Year: 2011
Type: article
Abstract: HOLZAPFEL, GENEVIEVE ANNE. Structural Analysis of Active Site Conformations in Ras and Rap1 GTPases. (Under the direction of Carla Mattos).Ras and Rap1 are monomeric GTPases in the Ras subfamily of the Ras superfamily that despite high sequence homology, have distinct diverse functions in the cell including involvement in cell proliferation and cell adhesion.GTPases undergo conformational changes associated with the transition from GTP to GDP predominately in the switch I and switch II regions.Switch I and switch II are dynamic regions that can take many different conformations.Here we examine the effect that small molecules can have on the equilibrium between different switch II conformations.We show that binding of calcium acetate in Ras to an allosteric site results in a catalytically active conformation that we call "allosteric on".We also show that binding of DTT or DTE in the interlobal region result in a different Ras catalytically inactive conformation we call "allosteric off".This conformation was also seen in oncogenic Ras mutants.The intrinsic hydrolysis mechanism of Ras is currently debated.Based on the allosteric on structure we propose a two water model.Our proposed mechanism can be supported by examining the protonation state of the active site residues.We have pursued neutron crystallography as a means to visualize hydrogen positions in Ras to support our hypothesis of intrinsic hydrolysis.Deuteration and perdeuteration are utilized in neutron crystallography to improve the signal to noise ratio in the neutron data collected.Resulting crystals were grown to a large volume and when mounted on a neutron beam diffraction spots were detected.This proves feasibility of collecting neutron diffraction data on Ras.Rap1, as the closest homologue to Ras in the Ras superfamily, provides a comparison protein for specificity determinants in Ras.The structure of Rap1 alone has not been solved.Additionally, specific analysis of Rap1 small molecule binding has also not been shown.The structure of Rap1 bound to GTP and GppNHp are also presented.These results show novel active site conformations in Rap1.The resulting active site conformations provide comparison to Ras with both differences and similarities.Furthermore, data are presented that examine small molecule binding to Rap1 using the Multiple Solvent Crystal Structures (MSCS) technique.Combined with the MSCS data of Ras, these data can disambiguate the subtle differences between Ras and Rap1.
Author: Genevieve Holzapfel
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2272785758