Site-directed mutagenesis of the ncd microtubule motor protein

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dc.contributor.authorSchmidt, William Richarden
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T21:52:41Zen
dc.date.adate2008-12-30en
dc.date.available2014-03-14T21:52:41Zen
dc.date.issued1996en
dc.date.rdate2008-12-30en
dc.date.sdate2008-12-30en
dc.description.abstractNcd is a member of the kinesin family of motor proteins. Ncd is involved in the processes of meiosis and early mitosis in <i>D. melanogaster</i>. PCR-mediated site-directed mutagenesis was utilized to introduce specific mutations into pET/MC6, a construct containing the motor domain of ncd. Six mutations were generated, two at glutamic acid residue 656, two at proline residue 649, one at arginine residue 623, and one double mutant at arginine residue 623 and threonine residue 632. Mutants proteins were expressed in bacteria and further characterized. Mutagenesis of the proline or glutamic acid residues resulted in insoluble proteins. The one exception is the mutagenesis of glutamic acid residue 656 into a glutamine, which resulted in a partially soluble protein. Mutagenesis of the arginine residue into an alanine (MC6-A623) resulted in a soluble protein while the double mutation of the arginine and threonine was insoluble. MC6-A623 exhibited a similar S-sepharose ion exchange chromatography binding and elution profile as MC6. Peptide antibodies made to conserved ncd motor domain sequences also recognized MC6- A623. The affinity of MC6-A623 (under the conditions tested) for microtubules was less than MC6. Most interestingly, under the conditions tested, MC6-A623 did not exhibit an increased ATPase rate in the presence of microtubules, a hallmark of the kinesin family of microtubule motor proteins. Analysis of the published ncd crystal structure, other motor protein sequences, and the experimental results of the mutagenesis of arginine residue 623, suggest that this residue is involved in the binding of MC6 to microtubules.en
dc.description.degreeMaster of Scienceen
dc.format.extentviii, 86 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-12302008-063348en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12302008-063348/en
dc.identifier.urihttp://hdl.handle.net/10919/46443en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V855_1996.S365.pdfen
dc.relation.isformatofOCLC# 36516396en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectkinesinen
dc.subjectmotor proteinen
dc.subjectncden
dc.subjectnon-claret disjunctionalen
dc.subjectsite-directed mutagenesisen
dc.subject.lccLD5655.V855 1996.S365en
dc.titleSite-directed mutagenesis of the ncd microtubule motor proteinen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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