23 January 2017

New tricks for old methods: STD NMR

According to our last poll, ligand-detected NMR is the most popular method for finding fragments. And among the several ligand-detected NMR techniques, the most popular appears to be saturation transfer difference (STD) NMR. The basic concept behind this approach is to selectively irradiate a protein, which then transfers its magnetization to any bound ligand, thus “saturating” (reducing the signals) for the ligand. Subtracting this spectrum from a reference spectrum reveals which ligand (if a mixture) or individual protons within a ligand are in close proximity to the protein.

Although STD NMR is fast and easy to run, it does have drawbacks. One is the fact that it requires pure protein: if there are other proteins in solution, it will be impossible to tell whether the small molecule binds to the protein of interest or to something else. This shortcoming has been overcome in a paper published recently in J. Biomol. NMR by Tamas Martinek and collaborators at the University of Szeged, the Hungarian Academy of Sciences, and the University of Debrecen.

In a normal STD experiment, the protein protons that are irradiated are far upfield (often around -0.5 ppm) – a region not relevant to most small molecules. These protons then transfer the magnetization throughout the protein and ultimately to any bound small molecules. In order to choose a specific protein, the researchers add an 15N-labeled antibody selective for the protein. They can then selectively irradiate the 15N-labeled antibody, which transfers its magnetization to the bound protein and from there to any bound ligand. They call this approach monoclonal antibody-relayed 15N-group-selective STD, or mAb-relayed 15N-GS STD.

To demonstrate the approach, the researchers observed the binding of 2 mM lactose to galectin-1 (Gal-1) using an 15N-labeled antibody against Gal-1. Lactose binds to Gal-1 with a dissociation constant of 0.155 mM, which is a relevant affinity for fragment screening. Gal-1 was present at 20 µM and the antibody was present at 10 µM, both of which are reasonably low. Control experiments established that both Gal-1 and the antibody were necessary, and the experiment was successful even in a cell extract.

So, as Teddy would ask, is this approach practical? You need an 15N-labeled antibody against your target, and it is important that the antibody is specific and does not compete with your ligand (ie, that it is non-neutralizing). Also, the amount of time required to acquire the spectra appears to be more than an hour. If this could be reduced, would 15N-GS STD assume a useful niche in the NMR toolbox?


viking_fury said...

So, the protein of interest is "dirty", but the antibody is 100% pure, 100% specific, does not compete for the binding site, and does not bind any small molecules itself. Then one only needs to run the experiments much longer, and STD experiments are officially "fixed" and free of artifacts. Right...
Great job driving down the impact factor of JBNMR!

Brendan said...

I have to admit, I haven't read the paper, but from your post it would seem to me that they are trading a technique with an issue of impure protein for a technique with multiple proteins. So instead of 1 protein which must be pure, you now have a 2 protein system in which you have to identify, label, express and purify an antibody instead of a pure protein, as well as ensure it is not competing/interfering with the ligand binding. In which case, you simply trade the issue of pure protein for a more complex system, in which you have more materials which can cause issues, labeling, twice the purification, specificity, competition, extended run time...

Surely one could use an orthogonal technique to weed out false positives instead.

Unknown said...

Do you really need a 100% pure target protein in the first place?

I thought in a 20 uM protein: 1 mM ligand system, it is OK if the protein is 95% pure. Assuming that 5% impurity does bind to the ligand, the impurity:ligand ratio would be 1000:1, for which I don't think significant STD effect would be present.