Topological examination of the bacteriophage lambda S holin by EPR spectroscopy.

The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2.