An engineered disulfide bridge in the transmembrane region of phage M13 coat protein stabilizes the alpha-helical dimer.
A single Cys-residue (Cys24) was introduced into the 50-amino acid major coat protein of M13 bacteriophage as part of a two-site substitution (Y24C-V31A) within the effective transmembrane (TM) segment (Tyr21 to Ile39) of the coat protein. Mutant Y24C-V31A was able to complete the phage life cycle and was shown to contain free sulfhydryls in the intact virus, as evidenced by susceptibility of Y24C-V31A phage to alkylation by Cys-specific 14C-iodoacetamide (14C-IAN). In contrast, the protein solubilized in deoxycholate micelles was resistant to 14C-IAN modification and was virtually inert to a transition from a characteristic alpha-helical oligomeric state to an aggregated beta-sheet structure relative to WT and V31A coat proteins, as shown by circular dichroism spectroscopy and SDS-PAGE. Reduction of mainly dimeric Y24C-V31A protein using beta-mercaptoethanol (beta-ME) generated monomeric species and resulted in a loss of helical thermostability. The overall results indicated that solubilization of Y24C-V31A coat protein into micelles resulted in formation of thermostable disulfide-bridged helical dimers. The disulfide bridge is deduced to be positioned along the stripe of residues involved in hydrophobic packing of TM parallel helical dimers.
Khan
A R
AR
Division of Biochemistry Research, Research Institute, Hospital for Sick Children, Toronto, Canada.
Deber
C M
CM
D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002250:Carbon Radioisotopes; D002942:Circular Dichroism; D016384:Consensus Sequence; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D007460:Iodoacetamide; D046911:Macromolecular Substances; D008823:Micelles; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D002250:Carbon Radioisotopes; D004220:Disulfides; D046911:Macromolecular Substances; D008823:Micelles; D011994:Recombinant Proteins; D007460:Iodoacetamide