I have been studying a dynamic protein and have selectively labelled a cysteine for 19F NMR. This cysteine has two peaks corresponding to a major and minor conformation. On a 500-MHz spectrometer, the relative populations are roughly 4:1. After I switched to a 700-MHz spectrometer, the relative populations are now roughly 2:1. I have confirmed that this is not a one-time fluke. Can a higher operating frequency enhance the minor population with respect to the major population?
I don't think the operating frequency (i.e. applied magnetic field) could cause a change in populations.
I think it is more likely to be caused by the difference in relaxation at different field strength, or how you see the change from one conformation to another.
Are there any differences in the peak shapes?
Hello,
Have you check the temperature calibration on both spectrometers? What type experiment have you done ? How have you estimated these ratios?
Sincerely,
Elisa
Jani Rahkila It doesn't look like there's a noticeable difference in line shape. I've attached a file of the spectrum at 500 MHz (blue) and at 700 MHz (red). Temperature is the same. Concentrations are similar.
Elisa Rioual I've double checked temperature. I've even run the spectra at different temperatures to see it that changes anything. It doesn't affect the relative populations. I'm running a 1D 19F NMR on a fluorine-tagged protein.
Have you calibrate your temperature on both spectrometers with methanol for example? (see this for example : http://staff.ustc.edu.cn/~liuyz/methods/NMR_VT_calibration.htm)
The diplaied temperature may not be the real temperature of your sample. You may have to callibrate it before running your experiment.
Have you try to put the same sample in both spectrometers ?
William Ford I don't think integrating those peaks (especially without using some deconvolution or line shape fitting) will give any meaningful values, especially for the blue spectrum. The S/N is simply too low.
Is the sharper peak some standard? The intensities of the broader peaks seem overall lower in the blue spectrum compared to the red. This could be to different relaxation at different fields, or due to molecular dynamics.
How long are the experiments? Would it be feasible to increase ns by a factor of 4 or 16 to get better sensitivity?
Dear Jani, it could also be a result of different relaxation effects depending on the magnetic field strengths. If one species is not relaxing completely to equilibrium magnetization, you can get partial saturation and the signal gets smaller. I am not familiar with 19F relaxation times, but to be absolutely sure, I would recommend to try a relaxation delay of 20 sec and see if there is a difference. With best regards, Mario
Elisa Rioual I'll ask the new instrument manager about a temperature calibration. I have tested the same sample on both spectrometers. It's definitely a variation by instrument. I've also tested different batches.
Jani Rahkila The left-most peak is the major conformation of a fluorine-tagged cysteine residue on my protein. The right-most peak is a minor conformation. (The little on in the middle is a different residue). I'm not concerned with integration at this point. The blue spectrum is probably broader because it's the 500 MHz. The 700 MHz gives sharper peaks.
I've never heard of field-dependent relaxation. Could you point me to some literature on that?
The experiments are roughly a half-hour. I've run different NS before, but I've never seen the relative intensities change.
Mario Schubert I've never considered playing with the relaxation times. I suppose I could give it a try.
Dear William,
Here is a good website explaining the T1 dependence with magnetic field and including references.
https://mriquestions.com/bo-effect-on-t1--t2.html
It focuses on 1H, but gives a good general idea.
Best,
Maxime
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