| Abstract
| - 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 Streptococcuspneumoniae, demonstrating that this approach can significantlyreduce the time required to determine protein structures by NMR.
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