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Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi–15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9459-z) contains supplementary material, which is available to authorized users.

In recent years, there has been a growing interest in fast acquisition and analysis of nuclear magnetic resonance (NMR) spectroscopy data for high throughput protein structure determination. Towards this end, rapid data collection techniques and methods to simplify the NMR spectrum such as amino acid selective unlabeling have been proposed recently. Combining these two approaches can speed up further the structure determination process. Based on this idea, we present three new two-dimensional (2D) NMR experiments, which together provide 15N, 1HN, 13Cα, 13Cβ, 13C′ chemical shifts for amino acid residues which are immediate C-terminal neighbors (i + 1) of residues that are selectively unlabeled. These experiments have high sensitivity and can be acquired rapidly using the methodology of G-matrix Fourier transform (GFT) NMR spectroscopy combined with non-uniform sampling (NUS). This is a first study involving the application of fast NMR methods to proteins samples prepared using a specific labeling scheme. Taken together, this opens up new avenues to using the method of selective unlabeling for rapid resonance assignment of proteins.