(S)Pinning down protein interactions by NMR

Abstract

Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions. These types of analyses determine the amounts and proportions of individual constituents that participate in a reaction as well as their rates of reactions and their thermodynamics. Although many different methods are available, there is currently no single method able to quantitatively capture and describe all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates, and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative descriptions of protein interactions. In this review we provide an easy‐access approach to NMR for the non‐NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background and illustrate simple protein–ligand interactions as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction.

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

“…47 In these titrations, changes in chemical shift are monitored as a function of protein-ligand ratios. For weak binding, the interaction is under the fast exchange regime ( k ex >> |Δω|) where k ex is the exchange rate of the interaction and Δω is the resonance frequency difference between the bound and free states.…”

“…The equation to determine the binding affinity values (K D ) was thus modified so that the denominator now contains [ L 0 ] instead of the more typical [P o ]. 47, 48 $${\Delta }_{obs}={\Delta }_{\max }\ast \frac{\left(K_D+\left[L_0\right]+\left[P_0\right]\right)-\sqrt{{\left(K_D+\left[L_0\right]+\left[P_0\right]\right)}^2-\left(4\left[p_0\right]\left[L_0\right]\right)}}{2\left[L_0\right]}$$ NMR titrations were performed at least in triplicate to determine K D values involving plasma derived ProT and thrombin. Duplicate titrations were carried out for the thrombin R77aA.…”

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data

“…Over the last two decades, the information that can be extracted from NMR observables has continuously increased owing to the development of sophisticated NMR methods combined with molecular biology protocols for sample preparation, which together have considerably enriched the NMR spectroscopist’s toolkit. The advent of these advances places NMR in the front line as one of the most valuable techniques to investigate complex protein systems under a multitude of conditions (Kay 2016 ; Sormanni et al 2017 ), which include the mapping of ligand binding sites and their affinities (Arai et al 2012 ; Teilum et al 2017 ), the presence of molecular crowders (Diniz et al 2017 ), different chemical compositions of the solution (Sengupta et al 2017 ) as well as site-directed mutations (Arbesú et al 2017 ) and local post-translational modifications (Theillet et al 2012 ) whose effects expand to the whole molecule. Such experimental design generates large and multivariable sets of NMR data.…”

Structural insight into proline cis/trans isomerization of unfolded proteins catalyzed by the trigger factor chaperone

“…Given the relatively weak affinity between the MBP fragment and TF as expected from ITC data ( Fig. S3 ) and the fact that the peak intensity change was monitored by the addition of substoichiometric amount of TF proteins, more significant intensity reduction, thus more significant line broadening, is attributed to slower binding kinetics, which tends to occur in stronger interaction ( 23 ) and/or higher population of the bound form. In either case, the extent of the peak broadening can be interpreted as an indication of the binding preference.…”