Laser cross-linking of proteins to nucleic acids. I. Examining physical parameters of protein-nucleic acid complexes.
Pulsed laser cross-linking results in efficient and rapid formation of covalent bonds between proteins and the nucleic acids to which they are bound, creating a "snapshot" of the protein-nucleic acid equilibrium existing at the moment of irradiation. The "frozen" equilibrium allows the determination of protein-nucleic acid binding constants, confirming both theoretical predictions and experimental determinations by standard physical chemical methods. Laser cross-linking results accurately reflect the alteration of protein-nucleic acid interactions induced by traditional methods such as increasing the salt concentration or by the addition of a nucleic acid that competes for binding of the protein. Thus this technique is very useful for the study of the association of proteins and protein complexes with nucleic acids under environmental conditions at which the reactions are not amenable to study by traditional physical chemical methods. In this paper we continue our calibration of the method, focusing primarily on interactions with single-stranded DNA-binding proteins and describe techniques for measuring quantitative interactions between nucleic acid constructs and single-protein or multiprotein complexes. Laser cross-linking can also provide direct evidence that binding correlates with functional activity.
Hockensmith
J W
JW
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Kubasek
W L
WL
Vorachek
W R
WR
von Hippel
P H
PH
D001426:Bacterial Proteins / Q000737:chemistry*; D017122:Bacteriophage T4 / Q000737:chemistry; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D007834:Lasers; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins