Journal of Biomolecular NMR

, Volume 53, Issue 3, pp 257–270 | Cite as

NMR line shapes and multi-state binding equilibria

  • Evgenii L. Kovrigin


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 ligand-binding 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 show that distinct multi-state 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 Kinetics Exchange Ligand binding Proteins 



A receptor


A ligand


Total concentrations of R and L, mol/L


A molar ratio of L and R in the sample serving as a marker of the titration progress


An equilibrium concentration of species X, mol/L


An equilibrium association constant for ligand binding


An equilibrium constant for intramolecular isomerization of the receptor (R ⇔ R*)


An equilibrium dimerization constant of the receptor (R ⇔ R2)



The author is deeply indebted to Dr. James Kempf for innumerable corrections, suggestions and comments, and Dr. Marius Clore for critical reading of the manuscript. The author acknowledges Dr. Mark Foster, Dr. Linda Nicholson, Dr. Brian Volkman, Casey O’Connor and Ian Kleckner for helpful discussions and practical comments. The author acknowledges Snehal Patil for creating the web interface for the LineShapeKin Simulation software and the Marquette University Committee on Research for financial support of web design (2012 Regular Research Grant).

Supplementary material (391 kb)
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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Department of ChemistryMarquette UniversityMilwaukeeUSA

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