NMR Methods for the Determination of Protein- Ligand Dissociation Constants

Abstract

This article is a review with 83 references of the application of NMR to the measurement of the dissociation constants of protein-ligand complexes. After briefly discussing some general concepts of molecular stability, the text turns to consider which NMR parameters are reporters of complex formation. The available data treatments required to translate observed NMR effects into quantitative measurements of the stability of the complex in the form of the dissociation constant (KD) are introduced. Linearisation methods and curve fitting methods are explained in detail and are illustrated with examples drawn from recent reports of protein-small molecule interactions. Throughout the text examples of the commonly observed NMR parameters , 1 / T1 and 1 / T2 are drawn from biological studies of 1H, 31P, 19F 15N (and other nuclei). The advantages of NMR diffusion experiments as a measure of KD are considered. Some less frequently used NMR approaches, some new ideas and some non-general methods are grouped together in a miscellaneous section. The major sources of errors in the determination of KD are identified. This allows recommendations for optimal experimental set up. Options for dealing with strong binding are reviewed. Finally, the implications of abstracting KD data from high throughput screening experiments are considered and several different approaches to generate this data are discussed.

Protein-Inhibitor Interaction Studies Using NMR

“…To generalize the description, we use the words “protein” and “ligand” in this section. These sets of molecules can be either an enzyme and an inhibitor, or a protein and a co-factor, or any system in which a protein interacts with a small molecule [79–81]. …”

The Arabidopsis Histone Methyltransferase SUVR4 Binds Ubiquitin via a Domain with a Four-Helix Bundle Structure

“…The 1 H and 15 N chemical shifts changes in the S4WIYLD domain were monitored via the two-dimensional 1 H– 15 N HSQC spectrum. The changes of amide 1 H and 15 N chemical shifts were averaged by using the formula 38 $$\mathrm{normal\Delta }{\delta }_{\text{av}}={\left\{\frac{1}{2}true[{(\mathrm{\Delta }{\delta }_{\mathrm{normalH}})}^2+{(0.2\mathrm{\Delta }{\delta }_{\mathrm{normalN}})}^{2}true]\right\}}^{1/2}$$The dissociation constant ( K d ) was determined from the changes in chemical shifts upon addition of S4WIYLD to ubiquitin by applying the formula 39 $$\mathrm{normal\Delta }{\delta }_{\text{av}}=\mathrm{normal\Delta }{\delta }_{\text{av,}max}\left[K_{\mathrm{d}}+{false[\mathrm{normalL}false]}_0+{false[\mathrm{normalP}false]}_0-\sqrt{{false(K_{\mathrm{d}}+{false[\mathrm{normalL}false]}_0+{false[\mathrm{normalP}false]}_{0}false)}^2-4{false[\mathrm{normalL}false]}_0{false[\mathrm{normalP}false]}_0}\right]/2false[\mathrm{normalP}false]$$where [P] 0 and [L] 0 are the concentrations of the protein and ligand, respectively, and Δ δ av,max is the maximal change in chemical shift that can be realized upon addition of ligand to protein. K d and Δ δ av,max were free parameters during the fit to the experimental data.…”

Small Molecule Inhibition of the Na+/H+ Exchange Regulatory Factor 1 and Parathyroid Hormone 1 Receptor Interaction

“…24 where Δ obs is the observed change in the chemical shift, Δ max is the chemical shift when the protein is completely saturated with ligand, L is the ligand concentration, and P is the protein concentration.…”