Three-Dimensional 13C-Detected CH3-TOCSY Using Selectively Protonated Proteins: Facile Methyl Resonance Assignment and Protein Structure Determination

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• 作者：John B. Jordan ; Helena Kovacs ; Yuefeng Wang ; Mehdi Mobli ; Rensheng Luo ; Clemens Anklin ; Jeffrey C. Hoch ; and Richard W. Kriwacki
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：July 19, 2006
• 年：2006
• 卷：128
• 期：28
• 页码：9119 - 9128
• 全文大小：1582K
• 年卷期：v.128,no.28(July 19, 2006)
• ISSN：1520-5126

文摘

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100kDa in size to be studied in detail using NMR spectroscopy. Using ²H/¹³C/¹⁵N enrichment and selectivemethyl protonation, we show that newly developed ¹³C direct detection methods can be used to rapidlyyield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. Wepresent a highly sensitive ¹³C-detected CH₃-TOCSY experiment that, in combination with standard ¹H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonatedresidues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner,K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.;Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin.Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared totheir fully protonated counterparts, allows significant simplification of spectra, and facilitates NOEassignments. Here, we demonstrate the usefulness of the ¹³C-detected CH₃-TOCSY experiment throughstudies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yieldshighly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) atlow concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile,Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediatesthe pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantlyreduce the time required to determine protein structures by NMR.