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dc.contributor.advisorYoung, Ryland F.en_US
dc.creatorPang, Tingen_US
dc.date.accessioned2011-08-08T22:47:45Zen_US
dc.date.accessioned2011-08-09T01:33:31Z
dc.date.available2011-08-08T22:47:45Zen_US
dc.date.available2011-08-09T01:33:31Z
dc.date.created2010-05en_US
dc.date.issued2011-08-08en_US
dc.date.submittedMay 2010en_US
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7781en_US
dc.description.abstractLysis of the host by bacteriophage 21 requires two proteins: the pinholin S21 (forms pinholes in the cytoplasmic membrane and controls lysis timing) and the endolysin (degrades the cell wall). S21 has a dual-start motif, encoding a holin, S2168, and a weak antiholin, S2171. Both proteins have two transmembrane domains (TMD) and adopt an N-in, C-in topology. The topology of S2168 is dynamic because TMD1 is a signal-anchor-release (SAR) domain which, while initially integrated into the cytoplasmic membrane, is eventually released into the periplasm. TMD1 is dispensable because the truncated protein, S2168?TMD1, retains the holin function. Adding two positive charges to N-terminus of S2168 by an irs tag (RYIRS) prevents the release of TMD1. The irsS2168 protein not only has lost its holin function, but is a potent antiholin and blocks the function of S2168. In this dissertation, the structure of S2168 was suggested by incorporating electron-microscopy, biochemical, and computational approaches. The results suggest that S2168 forms a symmetric heptamer, with the hydrophilic side of TMD2 lining the channel of ~ 15 A in diameter. This model also identifies two interacting surfaces, A and B, of TMD2. A model for the pinhole formation pathway was generated from analyzing phenotypes of an extensive collection of S21 mutants. In this model, the individually folded and inserted S21 molecules first form the inactive dimer, with the membrane-inserted TMD1 inhibiting the lethal function of TMD2 both inter- and intra-molecularly. A second inactive dimer may form, with one TMD1 released. When both TMD1s are released, the activated dimer is formed, with the homotypic interfaces A:A interaction of the TMD2s. However, this interaction might not be stable, which will shift to heterotypic A:B interactions, allowing TMD2 to oligomerize. Finally, the pinhole forms, possibly driven by the hydration of lumenal hydrophilic residues. In addition, the localization of pinholes was visualized by fusing the green fluorescent protein (GFP) to the C-terminus of pinholins. The results showed that pinholins form numerous small aggregates, designated as rafts, spread all over the cell body. The antiholin irsS2168 not only inhibits the triggering of S2168GFP, but inhibits the rafts formation as well.en_US
dc.format.mimetypeapplication/pdfen_US
dc.language.isoen_USen_US
dc.subjectbacteriophage 21en_US
dc.subjectholinen_US
dc.subjectlysisen_US
dc.subjectSAR domainen_US
dc.titleExploring the Pinhole: Biochemical and Genetic Studies on the Prototype Pinholin, S21en_US
dc.typeThesisen
thesis.degree.departmentBiochemistry and Biophysicsen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorTexas A&M Universityen_US
thesis.degree.nameDoctor of Philosophyen_US
thesis.degree.levelDoctoralen_US
dc.contributor.committeeMemberBryk, Maryen_US
dc.contributor.committeeMemberHolzenburg, Andreasen_US
dc.contributor.committeeMemberLi, Pingweien_US
dc.type.genrethesisen_US
dc.type.materialtexten_US


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