I am purifying a highly charged and disordered proteins and would like to solve its structure by NMR. The protein is soluble but at higher concentration and higher temperature, the favoured condition for NMR measurement, the protein slowly precipitated out of the solution. I now want to try buffer containing both L-Arginine and L-Glutamate which have been shown to prevent formation of salt bridges and therefore, protein aggregation. Do buffers containing these aa's would interfere with the NMR spectra analysis? Do you have also other suggestions what else to try in this case?
I never analyzed proteins myself but think that you will receive rather informative spectra if you can destinguish clearely signals of protein from the signals of buffer solvent. I see no no problem except overlapping of signals of
substrate and aminoacids of solvent
Yes, the protons from the buffer will dominate the NMR spectrum. Adjust the pH with DCl and NaOD to reach the maximum stability of your analyte.
if you are doing 15N experiments then no they don't interfere with the experiments. Even if you are doing 1H experiments, it might be ok if you don't have overlapping signals with the peaks you want to observe. However, you have to be careful with the concentration you use because if you add too much it will affect tuning and matching of the probe, similar to if you have high salt concentrations.
Thank for the answers
Stephen Boulton : will it somehow affect the 13C experiment? I am planning to use 50 mM of Arg and Glu each and 150 mM NaCl in the buffer
Dear Lam,
besides disturbing signals in 1H/13C (wich you migh minimize by use of deuterated amino acids) you will experience the problem that at 250 mM ionic strengh (which you plan) you will experience a substantial reduction of you detection sensitivity. If you are using a cryo-probe you will loose more than a factor of two compared to salt free conditions...
Alfred
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