Exploring the pinhole : biochemical and genetic studies on the prototype pinholin, S²¹ /
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| Other Authors: | |
| Format: | Thesis eBook |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University],
[2011]
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| Subjects: | |
| Online Access: | Link to OAK Trust copy |
| Abstract: | Lysis 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, S²¹68TMD1, 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. |
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| Item Description: | "Major Subject: Biochemistry" Title from author supplied metadata (automated record created 2011-08-09 15:09:44). Electronic resource. |
| Physical Description: | 1 online resource. |
| Bibliography: | Includes bibliographical references. |