VTechWorks staff will be away for the winter holidays starting Tuesday, December 24, 2024, through Wednesday, January 1, 2025, and will not be replying to requests during this time. Thank you for your patience, and happy holidays!
 

Identification of a protein kinase substrate in Sulfolobus solfataricus P2

Simple item page
dc.contributor.authorRedbird, Ruth Annen
dc.contributor.committeechairKennelly, Peter J.en
dc.contributor.committeememberHelm, Richard F.en
dc.contributor.committeememberPrater, Mary R.en
dc.contributor.committeememberMahaney, James M.en
dc.contributor.departmentBiochemistryen
dc.date.accessioned2014-03-14T20:09:42Zen
dc.date.adate2010-05-04en
dc.date.available2014-03-14T20:09:42Zen
dc.date.issued2010-04-01en
dc.date.rdate2012-04-12en
dc.date.sdate2010-04-15en
dc.description.abstractLiving organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies.en
dc.description.degreePh. D.en
dc.identifier.otheretd-04152010-143128en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-04152010-143128/en
dc.identifier.urihttp://hdl.handle.net/10919/26884en
dc.publisherVirginia Techen
dc.relation.haspartRedbird_RA_D_2010_Copyright.pdfen
dc.relation.haspartRedbird_RA_D_2010.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectArchaeaen
dc.subjectATP Synthaseen
dc.subjectphosphorylationen
dc.subjectprotein kinaseen
dc.titleIdentification of a protein kinase substrate in Sulfolobus solfataricus P2en
dc.typeDissertationen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

Files

Original bundle
Now showing 1 - 2 of 2
Thumbnail Image
Name:
Redbird_RA_D_2010_Copyright.pdf
Size:
2.51 MB
Format:
Adobe Portable Document Format
Download
Thumbnail Image
Name:
Redbird_RA_D_2010.pdf
Size:
4.95 MB
Format:
Adobe Portable Document Format
Download

Collections

Doctoral Dissertations