UNIVERSITY OF ILLINOIS
Grant: $7,150 - National Institutes of Health - Jun. 8, 2009
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Award Description: The purpose of the award was to support a pre-dent undergraduate or prematriculating dental student for a summer research experience. The student was expected to participte in an ongoing research project of a parent R01 grant that seeks to elucidate the molecular determinants of the serpin, protein Z-dependent protease inhibitor (ZPI), that are responsible for the ability of the serpin to inhibit membrane-bound factor Xa only when bound in a high affinity complex with protein Z. The studies that were pursued focused on engineering mutations in two sites of ZPI that were recently identified by our group and the group of Peter Gettins at UIC to mediate the interaction with protein Z by X-ray crystallography. The two ZPI interaction sites were proposed to be mutated independently, together and finally individual residues in the two sites wereto be mutated. The hypothesis to be tested was that the identified residues represent major contact sites for interaction of ZPI with protein Z in solution and these residues are the major contributors to the high affinity of the protein Z interaction.The student was expected to learn a number of research skills including mutagenesis, recombinant protein expression and purification, and functional assays for characterizing the mutant proteins including binding and kinetic assays that are routinely used in the laboratory. The student was expected to participate in all of the activities of the lab including weekly meetings of the Center for Structural Biology and to give presentations of results at our weekly lab meetings. The student was expected to gain valuable research experience that could impact his/her choice to pursue a future career path in biomedical research.
Project Description: The student, Jeff Gatti, was a pre-dent undergraduate who had not had any previous research experience, but had completed undergraduate course requirements for entry into dental school. Mr. Gatti assisted in the expression and purification of a number of mutant ZPI proteins which were designed to explore the role of residues implicated in the X-ray structure of the ZPI-protein Z complex in binding protein Z. Because two contact sites were observed in the crystal lattice, it was important to distinguish which contact sites reflected the true interaction site important for function and which represented the incidental contacts of the protein in the crystal. Mr. Gatti learned how to perform the mutagenesis of ZPI using synthetic mismatching oligonucleotides that targeted the site to be mutated and the polymerase chain reaction (PCR) to generate the desired mutant copies of the DNA and to package the DNA encoding ZPI into a virus that introduced the ZPI DNA into insect cells for the purpose of its expression. Mr. Gatti also assisted in the purification of the mutant proteins and characterizing their anticoagulant function. Specifically, Mr. Gatti sought to determine whether the mutations decreased the activating effect of protein Z on the ability of ZPI to inhibit membrane bound factor Xa. A mutant ZPI with four residues changed in one of the putative protein Z interaction sites was found to completely abolish protein Z activating function and the ability of protein Z to alter the electrophoretic mobility of ZPI. This finding provided proof of the functional site on ZPI for interaction with protein Z in the X-ray structure. Mr. Gatti demonstrated his knowledge of the scientific literature concerning ZPI anticoagulant function and made presentations of his results in our group lab meetings and a final presentation to all the students involved in the summer research program in the College of Dentistry.
Jobs Summary: The job created was that of a beginning research assistant and was intended for an undergraduate pre-dent or prematriculating dental student. The duties of the job were to assist in a research project supported by a parent NIH grant and the purpose was to gain a summer research experience that might influence future career goals of the student, such as pursuing a combined DDS/PhD degree or specialty training in biomedical research after the DDS. The student was expected to work with a senior postdoctoral associate in the lab to advance a research project intended to increase our understanding of the natural anticoagulant blood protein, protein Z-dependent protein inhibitor (ZPI), and its mechanism of protein Z and lipid-dependent regulation of blood coagulation factor Xa, the latter a key enzyme involved in activating the blood coagulation response to injury. The student was expected to learn a number of skills required to perform the research, to acquire knowledge of the current state of our understanding of ZPI and its anticoagulant function from the research literature and to present the results of work in our weekly lab meetings. The job provided earnings that covered basic living expenses of the student and provided education and training in research. (Total jobs reported: 1)
Project Status: More than 50% Completed
This award's data was last updated on Jun. 8, 2009. Help expand these official descriptions using the wiki below.
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