Chemical shift based editing of CH3 groups in fractionally 13C-labelled proteins using GFT (3, 2)D CT-HCCH-COSY: stereospecific assignments of CH3 groups of Val and Leu residues
- 111 Downloads
We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing 13C–1H cross peaks of CH3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the 13C–1H cross peaks of CH3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) 13C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) 13C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.
KeywordsIsotope labeling NMR Automated assignments Sequence specific resonance assignments Stereo-specific resonance assignments
The facilities provided by National Facility for High Field NMR at TIFR supported by Department of Science and Technology (DST), Department of Biotechnology (DBT), Council of Scientific and Industrial Research (CSIR), and Tata Institute of Fundamental Research, Mumbai, India, and NMR Research Centre at IISc supported by DST and are gratefully acknowledged. HSA acknowledges support from Department of Atomic Energy (DAE) BRNS and DST-SERC research awards. We thank Dr. Yogendra Sharma (CCMB, Hyderabad) for providing us the M-crystallin expression plasmid.
- Barnwal RP, Jobby MK, Sharma Y, Chary KVR (2006) NMR assignment of M-crystallin: a novel Ca2+ binding protein of the betagamma-crystallin superfamily from Methanosarcina acetivorans. J Biomol NMR 36 Suppl:1–32Google Scholar
- Chary KVR, Govil G (2008) NMR in biological systems: from molecules to humans. Springer, The Netherlands, pp 1–550Google Scholar
- Keller R (2004) The computer aided resonance assignment tutorial. Verlag GoldauGoogle Scholar
- Neri D, Szyperski T, Otting G, Senn H, Wüthrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28:7510–7516CrossRefGoogle Scholar
- Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552Google Scholar
- Tate S, Ushioda T, Utsunomiya-Tate N, Shibuya K, Ohyama Y, Nakano Y, Kaji H, Inagaki F, Samejima T, Kainosho M (1995) Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interactions between the N- and C-termini to maintain the inhibitory active form of cystatin A. Biochemistry 34:14637–14648CrossRefGoogle Scholar
- Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley Publishers, New-York, pp 1–292Google Scholar