Laser cross-linking of proteins to nucleic acids: photodegradation and alternative photoproducts of the bacteriophage T4 gene 32 protein.
Pulsed laser cross-linking provides a means of introducing a covalent bond between proteins and the nucleic acids to which they are bound. This rapid cross-linking effectively traps the equilibrium that exists at the moment of irradiation and thus allows examination of the protein-nucleic acid interactions that existed. Laser irradiation may also induce photodestruction of protein and we have used the bacteriophage T4 gene 32 protein to investigate this phenomenon. Our results show that both nonspecific and specific photoproducts can occur, specifically at wavelengths where the peptide backbone of proteins is known to absorb. These results demonstrate that nonspecific photodegradation can be correlated with the formation of a specific photodegradation product. The formation of this product was monitored to show that product yield is nonlinearly dependent on laser power and wavelength. We have also investigated an unexpected photoproduct whose formation is dependent on the length of the polynucleotide to which the gene 32 protein binds and that further demonstrates the complexities of analyzing protein-nucleic acid interactions through the use of UV laser cross-linking. These data provide essential information for the establishment of appropriate conditions for future studies that use UV cross-linking of protein-nucleic acid complexes.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
W L
WL
Kubasek
D
D
Spann
J W
JW
Hockensmith
D003432:Cross-Linking Reagents; D004279:DNA, Viral / Q000737:chemistry / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000528:radiation effects*; D007834:Lasers; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000528:radiation effects; D010777:Photochemistry; D014764:Viral Proteins / Q000737:chemistry / Q000528:radiation effects*
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4