Accuracy and precision of proteinā??ligand interaction kinetics determined from chemical shift titrations
Abstract NMR-monitored chemical shift titrations for the study of weak proteinā??ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (
K D ) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the
K D value of a 1:1 proteinā??ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125ā??138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of 1Hā??15N 2D HSQC NMR spectra acquired using precise proteinā??ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (
k off ). For experimentally determined kinetics in the fast exchange regime on the NMR timescale,
k off ~ 3,000 sā??1 in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for
k off from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise
k off values over a wide range, from 100 to 15,000 sā??1. The validity of line shape analysis for
k off values approaching intermediate exchange (~100 sā??1), may be facilitated by more accurate
K D measurements from NMR-monitored chemical shift titrations, for which the dependence of
K D on the chemical shift difference (Ī?
Ļ?) between free and bound states is extrapolated to Ī?
Ļ? = 0. The demonstrated accuracy and precision for
k off will be valuable for the interpretation of biological kinetics in weakly interacting proteinā??protein networks, where a small change in the magnitude of the underlying kinetics of a given pathway may lead to large changes in the associated downstream signaling cascade.
- Content Type Journal Article
- Category Article
- Pages 1-22
- DOI 10.1007/s10858-012-9678-6
- 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
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Accuracy and precision of proteinā??ligand interaction kinetics determined from chemical shift titrations
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