Take a theoretical protein of 200 residues for example: there should be 240 N-H HSQC correlations (considering ASN, GLN, and et al) on NMR spectrum.
However, due to the efficiency/efficacy of 15N labelling, not all nitrogen atoms in the protein are labelled.
So is it possible for the protein harvested from growth medium A to have 180 peaks on 1H-15N HSQC spectrum while that from growth medium B to have a different set of 180 peaks, and only 150 of these peaks are shared (overlapped) between A and B?
Thanks,
Yun
Hi there,
RMN spectrum results from a mixture of protein molecules therefore the non uniformity of the labeling will be averaged.
Expecting 240 peaks and having 180 can be considered normal. Maybe this can be of interest http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07381.x/abstract
Before thinking about the growth media, i'd check protein stability, NMR spectra quality (1D), presence of unfolding/aggregates, presence of proteolytic cleavage, presence of impurities/metals (if there is something bound to your protein, you may have a set of peaks shifting and that could justify the 30 peaks not overlapping).
You can even quantify the % of 15N labelling, but as Dominique Liger was saying, the non uniform labeling gets averaged, so if you have 50% instead of 90% labeling it should affect the entire spectra (overall reduction of signal/noise) and not only a few peaks.
Having less peaks doesn't mean necessarily non-uniform labeling.
1-It could be that the peaks that aren't seen are corresponding to area in the protein where there's exchange between two states on ms -us time scale which leads to peak broadening and loss of peaks.
2-It can be also that there are areas of the protein which are flexible and share similar chemical environment so what u think as one peak is indeed for 2 residues on top of each other due to similar chemical environment.
# I would try making the protein deuterated 15N labelled (this will reduce the relaxation due to dipolar coupling and see. Good luck
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