Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking.
Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.
Shamoo
Y
Y
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Williams
K R
KR
Konigsberg
W H
WH
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine / Q000032:analysis*; D010777:Photochemistry; D013604:T-Phages / Q000378:metabolism* / Q000528:radiation effects; D014764:Viral Proteins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C027903:oligo (dT); D010649:Phenylalanine