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What is meant by saturation transfer method in NMR? How it is useful in giving the structure of blue copper proteins like Azurin, Plastocyanin and Stellacyani? What is the need to go for this technique in blue copper proteins?

asked Aug 27 '10 at 06:17

santhosh%20kumar's gravatar image

santhosh kumar

updated Sep 05 '10 at 10:38

Evgeny%20Fadeev's gravatar image

Evgeny Fadeev

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Although I don't have any knowledge about 'blue copper proteins' vis-a-vis NMR, I will volunteer about the saturation transfer technique, in general . I think the method has become most popular within the last decade, due mainly to the publication by Mayer and Meyer ( JACS 2001, VOL.123, p 6108-6117), where they introduce the concept of 'epitope mapping' using saturation transfer difference method. Let us say, you have a small ligand molecule like a putative drug molecule and you want to know where does it bind on a large enzyme molecule and how effectively it binds. If you want to look at the enzyme using NMR to answer these question, you have two important steps to be fulfilled (a) you need to know the 3D structure of the enzyme (b) your enzyme should be observable with clarity, via NMR, at the possible site of binding, say a loop region or a cleft.

Both these steps are prohibitive if not impossible and most of the time we are interested in the second part of the question to begin with, i.e. how effectively does my drug molecule binds to the target. STD NMR (Saturation Transfer Difference NMR) is ideally suited for this. What more, you can throw in a bunch of small molecules that you think might be good candidates to be drug molecules to bind this particular enzyme and STD NMR can do a 'competition assay' and give the relative scores of which molecule binds the best.

Now to the technique. Please be forewarned that I am giving a rather hand waving description of this technique here : Let us consider only one type of a putative drug molecule for simplicity here. You dissolve the protein/enzyme/biomolecule in a suitable buffer and mix in the putative drug molecule at a N: 1 ratio to the protein, where N > 1 i.e. in excess (you have more than one drug molecule per enzyme molecule). Due to the large molecular weight of the protein the 1H spectrum is rather broad without any discernible features due to the strong coupling network of all the nuclei within the protein molecule. In contrast, the 1H spectrum of the small drug molecule will look like the usual high resolution NMR spectrum with well defined resonances from the different functional groups. To perform the STD experiment, you can select a frequency like 0 ppm and pre-saturate this point and due to spin-diffusion, the entire protein will be affected by the pre-saturating irradiation. Now if the putative drug molecule were to bind strongly at a given site on the protein, this saturation will transfer to this drug molecule also. If I record a 1H spectrum by applying a simple read pulse at the end of this pre-saturation, I will obtain the high-res 1H spectrum of the drug molecule, with the intensity of several resonances smaller than what they are in a fully relaxed condition. The difference in the intensity between the 'saturated 1H spectrum' of the drug molecule and its unsaturated i.e. control spectrum provides the qualitative information as to which part of the drug molecule effectively binds to the protein and also the quantitative information as to how strong this binding is.

I humbly remind myself that I have 'skimmed' through several important considerations that go with making the technique work properly. Nevertheless, if someone wants to take a first stab at it, probably this provides a casual introduction.


answered Aug 27 '10 at 07:51

DrSpin's gravatar image


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STD NMR is a powerful and fast method used to study protein-ligand interactions, focusing on the signals of the ligand. Besides, values including KD, the association and dissociation kinetics (Koff and Kon respectively) can be obtained from STD NMR study as well.

STD-NMR is a simple and fast method to screen ligand and characterize protein binding and it does not require expensive stable isotope or radioisotope labeling. Even to monitor low receptor concentration at low molecule concentration, STD can be used via non-scalar magnetization transfer, while NMR spectroscopy requires a much higher concentration of proteins that are labeled stable isotopes. In an STD-NMR experiment (as shown in the figure), the di?erence spectrum is obtained by subtracting a spectrum where the protein is selectively saturated (on-resonance) with signal intensities ISAT from that without saturation (off-resonance) with intensities I0. And the difference in intensities (ISTD=I0-ISAT) indicates molecule binding.

Creative Biostructure MagHelix™ Saturation Transfer Differences (STD) NMR, hope helpful.


answered Jun 29 '16 at 19:26

Justin%20Frank's gravatar image

Justin Frank

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