CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample

• Published in: Journal of biomolecular NMR 2015-12, Vol.63 (4), p.389-402
• Main Authors: Kerfah, Rime, Hamelin, Olivier, Boisbouvier, Jérôme, Marion, Dominique
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Biochemistry; Biochemistry, Molecular Biology; Biological and Medical Physics; Biophysics; Isoleucine - chemistry; Leucine - chemistry; Life Sciences; Malate Synthase - chemistry; Molecular Weight; Nuclear Magnetic Resonance, Biomolecular - methods; Physics; Physics and Astronomy; Spectroscopy/Spectrometry; Valine - chemistry
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-015-9998-4

A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13 C– 13 C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers. To permit the use of this efficient pulse scheme, a series of optimized precursors were designed to yield linear 13 C perdeuterated side-chains with a single protonated CH 3 group in these three residues. These precursors were added to the culture medium for incorporation into expressed proteins. For Val and Leu residues, the topologically different spin-systems introduced for the pro - R and pro - S methyl groups enable stereospecific assignment. All CH 3 can be simultaneously assigned on a single sample using a TOCSY experiment. It only requires the tuning of a mixing delay and is thus more versatile than the relayed COSY experiment. Enhanced resolution and sensi-tivity can be achieved by non-uniform sampling combined with the removal of the large J CC coupling by deconvolution prior to the processing by iterative soft thresholding. This strategy has been used on malate synthase G where a large percentage of the CH 3 groups could be correlated directly up to the backbone Ca. It is anticipated that this robust combined strategy can be routinely applied to large proteins.