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.
- Research Organization:
- Yale Univ., New Haven, CT (USA)
- OSTI ID:
- 6599544
- Journal Information:
- Proteins; (United States), Vol. 4:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PROTEINS
CROSS-LINKING
AMINO ACID SEQUENCE
BACTERIOPHAGES
GENETIC RADIATION EFFECTS
LIQUID COLUMN CHROMATOGRAPHY
NUCLEOTIDES
PHENYLALANINE
PHOTOCHEMISTRY
ULTRAVIOLET RADIATION
AMINO ACIDS
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
ELECTROMAGNETIC RADIATION
GENETIC EFFECTS
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
POLYMERIZATION
RADIATION EFFECTS
RADIATIONS
SEPARATION PROCESSES
VIRUSES
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture