Abstract
In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of “TROSY by isotope labeling” has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52–57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.
Highlights
The NMR spectra of larger proteins are characterized by numerous overlapped signals, which are severely broadened by dipolar interactions between nearby protons
It may not be necessary to gather all of the NMR signals for studying protein structures and dynamics, since the amino acid side-chains contain somewhat redundant structural information
The structural probes are presently limited to the transverse relaxation spectroscopy (TROSY) methyl and backbone amide signals for larger proteins (Pervushin 2000; Tugarinov et al 2003; Tugarinov and Kay 2003), in this article we show that any type of NMR signals other than those from methyl and amide groups; i.e., aromatic and aliphatic CH groups, can be observed well even for the 82 kDa single domain protein, malate synthase G (MSG), by optimizing their transverse relaxation properties by appropriate isotope labeling
Summary
The NMR spectra of larger proteins are characterized by numerous overlapped signals, which are severely broadened by dipolar interactions between nearby protons. The structural probes are presently limited to the TROSY methyl and backbone amide signals for larger proteins (Pervushin 2000; Tugarinov et al 2003; Tugarinov and Kay 2003), in this article we show that any type of NMR signals other than those from methyl and amide groups; i.e., aromatic and aliphatic CH groups, can be observed well even for the 82 kDa single domain protein, malate synthase G (MSG), by optimizing their transverse relaxation properties by appropriate isotope labeling.
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