title: Thermodynamic and functional characterization of protein W from bacteriophage lambda. The three C-terminal residues are critical for activity.
abstract: Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions. Composed 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 properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus. The purified wild type fusion proteins were found to be stably folded and biologically active. The protein 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, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C. Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also designed destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: K L
authors.initials: KL
authors.lastname: Maxwell
authors.forename: A R
authors.initials: AR
authors.lastname: Davidson
authors.forename: H
authors.initials: H
authors.lastname: Murialdo
authors.forename: M
authors.initials: M
authors.lastname: Gold
mesh_terms: D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014461:Ultracentrifugation; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015678:Viral Structural Proteins; C411883:protein W, bacteriophage lambda; D006639:Histidine