Simplification of Protein NOESY Spectra Using Bioorganic Precursor Synthesis and NMR Spectral Editing

摘要

In the past decade, NMR spectroscopy has evolved into an efficient tool for structure determination of proteins in solution.1,2 Conventional NMR structure determination relies on a large number of nuclear Overhauser enhancement (NOE) restraints. Typically, a large number of restraints per residue (e.g., 15-20) is required to obtain three-dimensional protein structures of reasonable accuracies and precisions. Yet, in practice, the number of obtainable restraints is considerably smaller, predominantly due to severe spectral overlap and because of line broadening. A major advance in this regard has been the introduction of highly deuterated, 15N,13C-labeled protein samples which have attenuated transverse relaxation rates and lead to significant gains in sensitivity and spectral resolution. Another important improvement has been the development of the TROSY-detection scheme.5 Applications include the 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase,6 or NMR investigations of the 723-residue monomeric enzyme malate synthase G from E. coli (81.4 kDa).7,8 Recently, NMR methods development has focused on correlation spectroscopy for side chain methyl groups.9 First, methyl groups have favorable relaxation properties and thus give rise to intense correlation peaks.10 Second, given their location in hydrophobic cores of proteins and protein interaction interfaces, methyl NOEs are valuable sources for structural information. Third, there are robust techniques for the incorporation of protonated Ile, Leu, and Val methyl groups in highly deuterated ~(13)C,~(15)N-labeled proteins.12 Finally, they are excellent reporters of protein dynamics and provide unique insight into functionally relevant protein motions.