Thermodynamic and functional characterization of protein W from bacteriophage lambda - The three C-terminal residues are critical for activity

Gene product W (gpW), the head-tail joining protein from bacteriophage lamb da, provides a fascinating model for studying protein interactions. Compose d of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA. To study the structural and functional p roperties of gpW, plasmids were constructed expressing gpW with hexahistidi ne tag sequences at either the N or C terminus. The purified wild type fusi on proteins were found to be stably folded and biologically active. The pro tein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism. Circular dichroism studies indicate that the protein is 47% helical, with a T-m of 71.3 degrees C, an d a Delta G(u) of 3.01 kcal/mol at 25 degrees C, Mutagenesis of the three h ydrophobic C-terminal residues of gpW showed that they are critical for act ivity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also desi gned destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Es cherichia coli.