Purification and biochemical characterization of the lambda holin.
Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the lambda holin, and one of the two products of gene S is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated S105H94, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an S mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.
Smith
D L
DL
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Struck
D K
DK
Scholtz
J M
JM
Young
R
R
D000595:Amino Acid Sequence; D001433:Bacteriolysis; D010582:Bacteriophage lambda / Q000254:growth & development*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006639:Histidine; D008081:Liposomes; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics; D010455:Peptides / Q000235:genetics; D010539:Permeability; D015202:Protein Engineering; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
D008081:Liposomes; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; C108624:S holin, bacteriophage lambda; D014764:Viral Proteins; C033223:polyhistidine; D006639:Histidine