Dewey, Jill Sayes (2010-08). Characterization of the Bacteriophage Lambda Holin and Its Membrane Lesion. Doctoral Dissertation. Thesis uri icon

abstract

  • 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.
  • 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.

publication date

  • August 2010