I have been analyzing 90-amino-acid fragment of a protein using HSQC and triple-resonance (1H-15N-13C) NMR. I know roughly where arginine, lysine, and tryptophan side-chain peaks lie on the HSQC, but how can I definitely distinguish what is a backbone and what is a side-chain? In addition to the HSQC, I have several 3D experiments (e.g. CBCANH and CACB(CO)NH).
Arg Nε-Hε and at low pH Arg Nη-Hη and Lys Nζ-Hζ are visible in HSQC but these are aliased/folded peaks. Go for an open Sweep Width (in 15N dimension) HSQC where these peaks will appear at their exact 15N ppm values and you can identify them easily. If you go to the R/K side chains' 15N plane in CBCANH, you will observe CD and CG of R or CE and CD of K. The Trp side-chain usually shifted downfield and appear near the bottom left corner.
Start with the assignment, you will understand which one is the backbone NH because in CBCANH and CACB(CO)NH they will show only the CA and CB (these you have to identify based on their C ppm values). But the side chains will be different.
Best wishes.
Side chains do not give cross-peaks in the Carbon 3D experiments like the one you mentioned (CBCACONH) so once you have done the peak-peaking of all HSQC you can extract the strips from the HNCA (for example): the strips corresponding to side-chains will not have observable cross-peaks.
Obviously there are exceptions as some residues being particularly flexible, hence you might not see the cross peaks for these residues, but for a 90 aa protein it should be quite straightforward.
You also easily identify by looking at the HSQC spectrum and see the two peaks at the same 15N ppm that you can draw a horizontal line at the top-right corner. If you already have 3D spectra, just run automated backbone assignments (http://i-pine.nmrfam.wisc.edu) and see what are left.
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