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What makes the lysis clock tick? A study of the bacteriophage holinWhite, Rebecca Lynn 15 May 2009 (has links)
The timing of host lysis is the only decision made in the bacteriophage lytic
cycle. To optimize timing, double-stranded DNA phages use a 2-component lysis
system consisting of a muralytic enzyme, the endolysin, and a small membrane protein,
the holin, which controls the timing of lysis. The best characterized holin gene to date is
the S gene of bacteriophage λ.
One unusual feature of the S gene is that it produces two proteins of opposing
function: the holin, S105, and the antiholin, S107. Raab et al isolated and characterized
a number of S mutants, but all of them expressed both the holin and the antiholin; it is
possible, then, that the true extent of the holin-holin interactions were masked by
interactions with the antiholin. Thus, a large number of S105 mutants were created, and
their phenotypes characterized in the absence of the antiholin. The interaction between
those mutants and the wild-type were examined in an attempt to better understand what
determines the timing of hole formation by S105.
S105 and S107 differ only by two amino acids at the N-terminus; S107 has an
additional Met-Lys sequence. Previous studies have shown that S107 may have a different topology to S105, where the N-terminus of S107 is located in the cytoplasm
and is cannot flip through the membrane because of the extra cationic side chain. This
study investigates the role of the N-terminal transmembrane domain of the S proteins in
terms of hole formation and its role in the antiholin character of S107.
Previous results suggest that S105 forms hole via a large oligomeric structure
termed the “death raft”. The death raft model states that after S105 is inserted into the
membrane, it forms “rafts”, which grow in size until a spontaneous channel forms
leading to depolarization of the membrane and hole formation. This study investigates
the pathway of hole formation at the single-cell level, using a C-terminal fusion of S105
and green fluorescent protein, and attempts to address several of the predictions posed
by the death raft model.
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Characterization of the Bacteriophage Lambda Holin and Its Membrane LesionDewey, Jill Sayes 2010 August 1900 (has links)
Bacteriophage holins are a diverse group of proteins that are responsible for the spontaneous and specifically-timed triggering of host cell lysis. The best-studied holin, S105 of phage lambda, is known to form lesions, or “holes”, in the inner membrane of E. coli which are large enough to allow the endolysin through to the periplasm. S105 has been studied extensively by both genetic and biochemical approaches; however, little is known about the mechanism of hole formation or the structure of the lambda holin and its inner membrane lesion.
An in vitro system for reconstituting hole formation by S105 was developed in which liposomes containing a self-quenched fluorophore served as artificial cell membranes (1-2). Upon delivery of solubilized S105 to the liposomes, an increase in fluorescence was observed, indicating that the fluorophore within the liposomes had escaped into the surrounding media via an S105-mediated hole in the membrane. This in vitro system, which has been optimized in this work, has been a valuable biochemical tool for analysis and reconstitution of the pathway to S105 hole formation in the cell membrane.
Due to the difficulty associated with over-expression and purification of toxic membrane proteins, there are no solved structures of bacteriophage holins. Sample preparation and experimental conditions for NMR spectroscopy were optimized and structural information about a lambda holin mutant protein was obtained. Specifically, micellar contacts of transmembrane domain regions versus water contacts of the C-terminal region, secondary structure, and backbone dynamics were determined.
Cryo-electron microscopy was used to visualize the inner membrane lesions formed by phage holins [lambda] S105, P2 Y, and T4 T. Therefore, the large holes initially seen in cells expressing S105 are not specific to the lambda holin, nor to class I holins. The S105 holes average ~340 nm (3), and are the largest membrane lesions ever observed in biology. They are stable at their original size, and are not localized to a specific region of the membrane. In addition, missense mutants of S105 were used to correlate hole size, protein accumulation, and lysis timing in a current model for the S105 hole formation pathway.
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Determining the Structural Dynamics and Topology of Canonical HOLIN-S05 Using EPR SpectroscopyPerera, Rehani Shinuka 11 June 2020 (has links)
No description available.
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