Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh, the EJ-1 phage holin.
Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major alpha-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of N-terminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.
Haro
Amparo
A
Insto de Quimica-Fisica Rocasolano, Consejo Superior de Investigaciones Cientificas, Madrid 28006, Spain.
Vélez
Marisela
M
Goormaghtigh
Erik
E
Lago
Santiago
S
Vázquez
Jesús
J
Andreu
David
D
Gasset
María
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001435:Bacteriophages / Q000378:metabolism*; D002942:Circular Dichroism; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D010455:Peptides; C108616:LytH protein, Staphylococcus aureus; D009238:N-Acetylmuramoyl-L-alanine Amidase