A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions.
We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.
Bujalowski
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Lohman
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D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008954:Models, Biological; D010957:Plasmids; D011072:Poly U / Q000378:metabolism*; D011485:Protein Binding
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D046911:Macromolecular Substances; D011072:Poly U