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
Re: Temperature calibration: methanol- vs protein-based,