Increased precision for analysis of proteinâ??ligand dissociation constants determined from chemical shift titrations
Abstract NMR is ideally suited for the analysis of proteinâ??protein and protein ligand interactions with dissociation constants ranging from ~2 ÎĽM to ~1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (
K D ) of 1:1 proteinâ??protein or proteinâ??ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the
K D , and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters
K D and the maximum chemical shift change (Î?δmax). During a typical NMR titration, the initial protein concentration, [
P 0], is held nearly constant. For this condition, to determine the most accurate parameters for
K D and Î?δmax from nonlinear least squares analyses requires initial protein concentrations that are ~0.5 Ă?
K D , and a maximum concentration for the ligand, or titrant, of ~10 Ă? [
P 0]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted
K D and Î?δmax values when [
P 0] >
K D . Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (
K D and Î?δmax) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 proteinâ??ligand interactions.
- Content Type Journal Article
- Category Article
- Pages 1-14
- DOI 10.1007/s10858-012-9630-9
- Authors
- Craig J. Markin, Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada
- Leo Spyracopoulos, Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada
Source: Journal of Biomolecular NMR