Affinity measurements in a single NMR tube?

Last week we highlighted a ligand-detected NMR method to measure affinities of protein-ligand interactions. That technique, R2KD, requires preparing multiple NMR samples with the ligand at different concentrations. In a new open-access paper published in J. Am. Chem. Soc., Serena Monaco and collaborators at University of East Anglia and Universidad de Sevilla describe a method that can be done in a single NMR tube.

 

The researchers have actually combined two methods, chemical shift imaging (CSI) and Saturation Transfer Difference (STD) NMR, to create imaging STD NMR. We’ve written previously about STD NMR, which relies on the transfer of magnetization from an irradiated protein to a bound ligand. In CSI, chemical shift information is recorded at multiple slices along the length of an NMR tube. Normally the solution in an NMR tube is homogenous and so the chemical shifts would be identical at the bottom and top of the NMR tube. Here, though, the researchers create concentration gradients by carefully pipetting a solution containing ligand on top of a solution containing protein and allowing the ligand to diffuse the length of the NMR tube.

 

Like all things NMR-related, the mathematics get a bit complicated. One important factor is the rate of diffusion for a given small molecule. This “diffusion coefficient” can be experimentally measured by creating a concentration gradient in the absence of protein and measuring the ligand concentration at various positions in an NMR tube after a given length of time (typically more than 12 hours). Diffusion is dependent on molecular weight, so it is also possible to calculate the diffusion coefficient, and in fact the researchers found that the calculated values matched the experimental values for three different small molecules.

 

Knowing the diffusion coefficient helps establish the maximum ligand concentration to use and the ideal diffusion time. The researchers examined three different protein-ligand pairs, all of which had weak affinities, with K_D values from 0.2 to 2 mM. Measuring STD signals at different slices along the NMR tube effectively yields STD signals at different concentrations of ligand, and fitting this to an equation allows calculation of the dissociation constant. For the three model systems the affinities agreed with literature values, which had been determined using ITC or WAC.

 

One nice feature of imaging STD NMR is that it can identify non-specific binding. This is because STD signals vary depending in part on how close a proton on the ligand is to the protein, resulting in different STD signals for different protons for specific binders. If this “epitope pattern” is lost at higher concentrations, this suggests non-specific binding, where the ligand can bind in random orientations to multiple sites on the protein. The researchers demonstrated this for one of their model systems: tryptophan binds specifically to bovine serum albumin with a dissociation constant of 0.2 mM, but above 1 mM or so the epitope disappears, suggesting non-specific binding.

 

Imaging STD NMR does have some limitations. For one thing, it requires a high initial concentration of ligand: 30 mM in the case of tryptophan, and even higher for the other two ligands. Most small molecules are nowhere near this soluble in water. The researchers suggest that ligands could be dissolved in DMSO and placed on the bottom of the NMR tube, with the protein solution gently layered on top. They show that the concentration gradients develop in a similar manner as a fully aqueous system, but acknowledge that high DMSO concentrations may not play well with most proteins.

 

Also not stated is the sensitivity of the method for higher affinity binders. Last week’s R2KD could measure affinities as tight as 10 µM, but it is unclear how much below 200 µM imaging STD NMR can go.

 

Finally, as we noted in 2019, STD effects are remarkably complex and not well-correlated with affinity. In particular, binding kinetics can play a role in the strength of the signal. It would have been nice to see more than three protein-ligand pairs tested.

 

All that said, this is an intriguing approach. Laudably, the researchers provide extensive supporting information, including mathematical derivation of the fitting equations, a spreadsheet, NMR pulse sequences, and macros. I’ll be curious to see how it works for others.