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Label-free NMR-based dissociation kinetics determination

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Abstract

Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly. Importantly, the use of large spin-lock fields allows the detection of faster dissociation rates than other relaxation approaches. Moreover, this experimental approach allows to access directly the off-rate of the binding process without the need for analyzing a series of samples with increasing ligand saturation. The validity of the method is shown with small molecule interactions using two macromolecules, bovine serum albumin and tubulin heterodimers.

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Funding

This work was supported by funds from the James Graham Brown Foundation, the National Center for Research Resources CoBRE (1P30GM106396), the Max Planck Society, and the EU (ERC Grant Agreement Number 233227 to C.G.). P.T.M. acknowledges the Humboldt Foundation for a postdoctoral research fellowship.

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Authors and Affiliations

  1. Department of NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Pablo Trigo-Mouriño & Christian Griesinger

  2. James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA

    Donghan Lee

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  1. Pablo Trigo-Mouriño
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  2. Christian Griesinger
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  3. Donghan Lee
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Correspondence to Donghan Lee.

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Trigo-Mouriño, P., Griesinger, C. & Lee, D. Label-free NMR-based dissociation kinetics determination. J Biomol NMR 69, 229–235 (2017). https://doi.org/10.1007/s10858-017-0150-5

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