Hi Edward!
On 08.01.2013, at 10:31, "Edward d'Auvergne" <edward@xxxxxxxxxxxxx> wrote:
For the temperature issues, maybe it is worth talking to the Bruker
people.
I will do that. I should have done it earlier, I guess.
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.
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.
For me, the spectra are perfectly identical. The only differences are
due to spectral noise shifting the peak randomly in height and
chemical shift in all directions equally.
Same here after I adjusted for the temperature via my TROSY/protein-based
method.
I would recommend ethylene glycol. Have a look at the published
literature on NMR temperature calibration and see what is best. Maybe
you need to modify the phases on your pulses to preserve the ethylene
glycol magnetisation but destroying the water and protein signals, and
then use the data from the recorded 2D for your calibrations. This
would avoid quick cooling between the experiments.
That's a good idea. I will look into this.
Thanks a lot,
Martin
Re: Temperature calibration: methanol- vs protein-based,