NMR line shapes and multi-state binding equilibria

Abstract

Biological function of proteins relies on conformational transitions and binding of specific ligands. Protein-ligand interactions are thermodynamically and kinetically coupled to conformational changes in protein structures as conceptualized by the models of pre-existing equilibria and induced fit. NMR spectroscopy is particularly sensitive to complex ligandbinding modes-NMR line-shape analysis can provide for thermodynamic and kinetic constants of ligand-binding equilibria with the site-specific resolution. However, broad use of line shape analysis is hampered by complexity of NMR line shapes in multi-state systems. To facilitate interpretation of such spectral patterns, I computationally explored systems where isomerization or dimerization of a protein (receptor) molecule is coupled to binding of a ligand. Through an extensive analysis of multiple exchange regimes for a family of three-state models, I identified signature features to guide an NMR experimentalist in recognizing specific interaction mechanisms. Results also show that distinct multistate models may produce very similar spectral patterns. I also discussed aggregation of a receptor as a possible source of spurious three-state line shapes and provided specific suggestions for complementary experiments that can ensure reliable mechanistic insight.

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

“…1). This pattern may be caused by four possible molecular mechanisms (Kovrigin 2012): (A) pre-existing dimerization equilibrium when the dimer is incapable of ligand binding, (B) dimerization of the bound state to form a dimer that cannot dissociate the ligands, (C) binding of two ligand molecules to different binding sites, and (D) the ligand binding followed by isomerization to a tightly bound complex (the induced fit mechanism) (Fig. 3).…”

“…3, panels a and d, to account for the experimental data by fitting of both mathematical models to a series of 1D spectral datasets of resonances of W28, G39, G153, and T157 resolved in proton dimension, and G43 and T157 resolved in the nitrogen dimension using IDAP NMR line shape analysis software (Kovrigin 2012). Results in the Fig.…”

“…5 cannot provide enough information for unambiguous determination of the product interaction mechanism (Kovrigin 2012). One of the means to reduce the conformational exchange rate constants without altering chemical structure of the protein is deuteration, which results in increased energy barriers for the hydrogen-bond making and breaking (Kohen 2003;Kovrigin and Loria 2006;Krantz et al 2000).…”

Resolving Three-State Ligand-Binding Mechanisms by Isothermal Titration Calorimetry: A Simulation Study

“…LineShapeKin Simulation was described in my previous report 8 and is freely available from http://lineshapekin.net. The models under discussion in this paper are given in Fig.…”

“…The ITC is frequently used to complement Nuclear Magnetic Resonance (NMR) studies to provide thermodynamic information on complex interaction mechanisms [5][6][7] . I previously reported analysis of NMR line shapes in titrations of systems with protein-ligand interactions coupled to isomerization or dimerization equlibria 8 . In this communication I am outlining major patterns one can expect in such three-state coupled equilibria from ITC experiments to help NMR and ITC experimentalists anticipate results from real systems.…”