Hi,
>> Every spectrometer behaves differently with respect to temperature, so >> temperature calibration is essential for any serious dynamics study. >> It is incorrect and dangerous to assume that the VT temperature >> setting is the same as the sample temperature. > > We're aware of that, that's why we calibrate the spectrometers regularly with > the help of our standard methanol sample. Our method just seems not that > accurate as I thought. I will try ethylene glycol. Note that different experiments add different amounts of power into the probe. So this calibration is ok for most standard experiments but is not suitable for high powered experiments (such as with intense spin lock periods, CPMG blocks, etc.) when temperature is important for you. >> https://gna.org/patch/download.php?file_id=16912 >> https://gna.org/patch/download.php?file_id=16913 >> >> From your spectra (the above file links), it looks suspiciously as >> though you have an exchange process occurring (though it may not be >> the case). Have you measured relaxation dispersion data for the >> system? > > All the dynamics stuff is pretty new to us and we just started implementing > the pulse schemes for the dispersion experiments. So, no, we didn't yet > measured relaxation dispersion, but it is definitely on the schedule. > > What makes you think there is exchange? In the pictures above only the > magenta/pink spectrum is a bit noisy (just above base plane level) and one > peak seems a bit displaced (~ 9.8/132.3 ppm, it's a Methionine). This residue > is not exposed to the solvent and also tightly bound in a beta-sheet. The > effect apparently is field-dependend, maybe due to ring currents from a > Histidine which are found close to the Met? > > But indeed – during the assignments of the proteins I noticed strips of > disappearing/broadened signals in my spectra. I have four different > complexes, not all of them show the same behavior in this respect. Some of > them pretty much have a complete set of signals, except for loop regions. At > least one protein has clearly a reduced set of signals. Also, some „double“ > peaks appear. I don't yet have a coherent picture of the dynamics of my > proteins, but this is why we started the project in the first place ;) Spin > relaxation seemed the best method to begin with, but there are still a lot of > things one has to take into account, one of them apparently perfectly > temperature-calibrated spectrometers. Just from the peak shapes, the differences in peak intensities, and the temperature shifts gives the impression of an exchange process. But this is just a hunch. This is also the Trp indole NH region of the spectra where I would expect all peaks to be equally intense due to nanosecond ring flips in the core and surface of the protein affecting the relaxation properties of the spin systems, resulting in strong peaks as you would expect from mobile bb sections of the protein. But you would need to confirm this. And if exchange is involved, then temperature is even more important ;) From the parts of the spectra you show, it looks like both standard relaxation and relaxation dispersion could possibly produce quite interesting results. Regards, Edward _______________________________________________ relax (http://www.nmr-relax.com) This is the relax-users mailing list [email protected] To unsubscribe from this list, get a password reminder, or change your subscription options, visit the list information page at https://mail.gna.org/listinfo/relax-users
