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Thursday, July 15, 2010

HSQC - TOCSY

There are many different NMR techniques used to probe molecular structure. Two very useful methods are the HSQC (or HMQC) sequence used to obtain heteronuclear coupling correlations and the TOCSY sequence used to look at extended homonuclear coupling correlations. These are among the most widely used pulse sequences by chemists to help elucidate the molecular structure of organic molecules. The sequences can also be combined into a single HSQC-TOCSY sequence which is simply an HSQC followed by a TOCSY spin lock. For the case of 1H and 13C in organic molecules, the HSQC-TOCSY spectrum provides 1H TOCSY subspectra of the isolated spin systems in the rows at each of the 13C frequencies. Overlap of 1H resonances which may have appeared in a simple 1H TOCSY spectrum can be resolved in the HSQC-TOCSY spectrum provided there is enough shift dispersion in the 13C spectrum. An example of this is shown in the figure below. The top panel shows the HSQC-TOCSY spectrum for 3-heptanone collected on a 300 MHz instrument. One can see the two spin systems on either side of the carbonyl group, color coded in yellow and pink. The second panel shows an expansion of the region within the red square of the first panel. Here we can separate the overlapping quartet and triplet for the methylene protons on either side of the carbonyl group.

2 comments:

Arifuzzaman Tapash said...

Hello sir,

I need your suggestion about the HSQC-TOCSY (or TOCSY-HSQC) experiment (on Varian Inova 600 MHz instrument).

As I’m a new user of Varian instrument and also new in protein NMR I'm facing some problems acquiring a 3D TOCSY-HSQC data of a membrane protein. In my current project I need to collect some 3D HSQC-TOCSY (or TOCSY-HSQC) data on Varian Inova 600 MHz instrument for protein side-chain assignment. My sample is a (1.2 m molar) membrane protein inside deuterated DPC micelle in D2O. Its a small membrane protein with MW (ca.) 4 kDa, and considering the micelle the total MW of the protein-micelle system should not exceed 20 kDa.


I was trying to use the “gtocsyNhsqc” (with water suppression) pulse sequence to collect the data. However, I couldn’t get any (usable) data using that pulse program, no cross-peak was seen. As the 3D pulse sequence wasn’t working, I ran a 2D ‘gtocsy’ experiment to see the proton-proton correlation but didn’t get any cross-peak also. The 1D data looks good, but in the 2D data, it looks like no polarization transfer is going on. I could not figure out the problem.

I tried (for the 2D gtocsy)-
>> Mixing time: 30 to 80 m sec
>> Relaxation delay: 1 to 2.5 sec
>> Spin-locking strength: 6000 (for 10 ppm sweep width)

(I can send you the pulse sequence for the 2D gtocsy and the result spectrum)


I found the same sample produces nice HSQC 2D spectrum, but for an unknown reason, I’m not getting any (tocsy) cross peak. As I’m new to this instrument and also new in protein NMR, I couldn’t figure out the problem yet. I don't think the problem is with the pulse program, I must doing something wrong in the parameter settings.


Could you help me to solve this problem? Any parameter which is important to consider? Any suggestion will be highly appreciated. If needed, I can provide more information.

Thanks!!!

Glenn Facey said...

Arifuzzaman,
I'm sorry. I am not an expert in 3D sequences for proteins on Varian instruments. I don' t think I could be of much help.
Glenn