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.
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3 comments:
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!
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.
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.
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