Application of the Multiple Solvent Crystal Structures Method to Analyze the Protein Binding Surface of H-Ras Protein

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dc.contributor.advisor Carla Mattos, Committee Chair en_US
dc.contributor.author Buhrman, Gregory Kale en_US
dc.date.accessioned 2010-04-02T19:17:30Z
dc.date.available 2010-04-02T19:17:30Z
dc.date.issued 2006-05-02 en_US
dc.identifier.other etd-05022005-064543 en_US
dc.identifier.uri http://www.lib.ncsu.edu/resolver/1840.16/5664
dc.description.abstract H-Ras is a member of the small, monomeric GTPase protein superfamily. H-Ras functions as a 'molecular switch', using nucleotide dependent conformational changes to relay signals in a number of signal transduction pathways. Mutations in codons 12, 13 and 61 creates an oncogenic version of the protein which does not hydrolyze GTP, resulting in the constitutive activation of downstream effector proteins. Ras proteins participate in multiple protein : protein interactions in the cell, making Ras a good candidate protein to extend the Multiple Solvent Crystal Structures method (MSCS) to the analysis and prediction of protein binding surfaces. MSCS involves solving the crystal structure of the protein after soaking the protein crystal in a variety of organic solvent molecules. Replacing an aqueous solvent with an organic solvent affects the Ras protein structure in several ways. The disordered Switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. Polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic co-solvents. This suggests that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals. We have used MSCS to study two crystal forms of active H-Ras bound to a nonhydrolyzable GTP analog (GMPPNP). We have also solved the structure of an oncogenic mutant of H-Ras (Q61L) in a non-canonical crystal form. This crystal form of H-Ras shows a new conformation for the flexible Switch II region that is not affected by crystal packing forces. This provides a structural explanation for the oncogenic properties of the Q61L mutation, showing that the Q61L mutation stabilizes a non-catalytic conformation of Switch II. MSCS analysis of Ras identifies the known Ras-effector binding domain as a site of protein: protein interaction and predicts a new protein binding site that is located in a large, solvent exposed pocket between Switch II and helix 3. In applying MSCS to the Ras protein, we show that by using polar organic solvent molecules as probes, we can identify binding sites that are highly charged and dynamic. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject protein structure en_US
dc.subject Ras en_US
dc.subject GTPase en_US
dc.subject organic solvents en_US
dc.subject MSCS en_US
dc.subject x-ray crystallography en_US
dc.subject protein binding sites en_US
dc.title Application of the Multiple Solvent Crystal Structures Method to Analyze the Protein Binding Surface of H-Ras Protein en_US
dc.degree.name PhD en_US
dc.degree.level dissertation en_US
dc.degree.discipline Biochemistry en_US


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