Abstract
Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. However, resonance assignment remains challenging. The problem is to combine existing structural informational with sparse distance restraints and search for the most compatible assignment among the permutations. Prior classification of peaks as either from isoleucine, leucine, or valine reduces the search space by many orders of magnitude. However, this is hindered by overlapped leucine and valine frequencies. In contrast, the nearest-neighbor nuclei, coupled to the methyl carbons, resonate in distinct frequency bands. Here, we develop a framework to imprint additional information about passively coupled resonances onto the observed peaks. This depends on simultaneously orchestrating closely spaced bands of resonances along different magnetization trajectories, using principles from control theory. For methyl-NMR, the method is implemented as a modification to the standard fingerprint spectrum (the 2D-HMQC). The amino acid type is immediately apparent in the fingerprint spectrum. There is no additional relaxation loss or an increase in experimental time. The method is validated on biologically relevant proteins. The idea of generating new spectral information using passive, adjacent resonances is applicable to other contexts in NMR spectroscopy.
Highlights
Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution
Developed computational techniques use inter-methyl distance restraints derived from nuclear overhauser effect (NOE) experiments, compared with distances extracted from the protein structure as a means to obtain resonance assignments for methyl residues[19,20,21,22,23,24,25,26]
We demonstrate that the leucine and valine signals can be clearly distinguished using a specially designed selective homonuclear decoupling pulse during the indirect 13C chemical shift encoding delay
Summary
Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. Developed computational techniques use inter-methyl distance restraints derived from nuclear overhauser effect (NOE) experiments, compared with distances extracted from the protein structure as a means to obtain resonance assignments for methyl residues[19,20,21,22,23,24,25,26]. One way to distinguish leucine from valine peaks in spectrum is by making two different samples using specialized precursors for selective labeling of leucine[27,28,29], which is costly and labor intensive, especially for membrane proteins. One can distinguish leucine from valine peaks by preparing an additional sample where valine methyl is labeled with NMR inactive 12C rendering those peaks absent from the spectrum This approach is slightly cheaper than one described before but still it is labor intensive. Extra delays result in severe relaxation losses, which can make these approaches unsuitable for large or challenging proteins
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