An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N,13C-laheled proteins

The Protein Engineering Centers of Excellence, University of Toronto, M5S 1A8, Toronto, ON, Canada.
Journal of Biomolecular NMR (Impact Factor: 3.14). 10/1996; 8(3):351-6. DOI: 10.1007/BF00410333
Source: PubMed


A biosynthetic strategy has recently been developed for the production of (15)N, (13)C, (2)H-labeled proteins using (1)H(3)C-pyruvate as the sole carbon source and D(2)O as the solvent. The methyl groups of Ala, Val, Leu and Ile (gamma2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a (15)N, (13)C, (2)H, (1)H(3)C-labeled SH2 domain at 3 degrees C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30-40 kDa molecular weight range.

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    • "Methyl-bearing side chains with 1 H– 13 C labeled methyl groups are listed in the second column a The five samples correspond to R1p conjugated to surface engineered cysteines at A52C, Q87C, S113C, S196C and A241C J Biomol NMR (2011) 51:319–328 321 of 18.7, 1.0, 18.7 and 1.0 ppm in the F 1 ( 13 C), F 2 ( 1 H), F 3 ( 13 C) and F 4 ( 1 H) dimensions, respectively (total duration 90 h). A 4D HC(CO)NH-TOCSY experiment (Gardner et al. 1996 "
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    ABSTRACT: Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.
    Full-text · Article · Sep 2011 · Journal of Biomolecular NMR
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    • "), (H)CCTOCSY(CO)NNH, and H(CC)TOCSY(CO)NNH (Montelione et al. 1992; Grzesiek et al. 1993; Logan et al. 1993; Lyons and Montelione 1993; Gardner et al. 1996 "
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    ABSTRACT: A key step in the signaling cascade responsible for activation of the transcription factor NF-kappaB involves Lys63-linked polyubiquitination of TRAF6. Covalent attachment of ubiquitin (Ub) to TRAF6, and subsequent poly(Ub) chain synthesis, is catalyzed by the hUev1a-hUbc13 heterodimer. hUbc13 is a catalytically competent E2 enzyme, and hUev1a is an E2-like protein that binds substrate Ub. The hUev1a-hUbc13 heterodimer is targeted to TRAF6 through interactions between hUbc13 and the N-terminal RING domain from TRAF6. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution state structure of the RING domain from human TRAF6, and the interaction between hUbc13 and TRAF6 was characterized using NMR chemical shift mapping. The main-chain dynamics of the RING domain from TRAF6 were studied using (15)N NMR relaxation. Analysis of the main-chain dynamics data indicates that residues within the alpha-helix and beta-sheet of the RING domain are as rigid as regions of canonical secondary structure in larger proteins, consistent with the biological role of RING-domain E3 proteins, which requires that the E3 contain a recognition site for recruitment of E2 ubiquitin conjugation enzymes.
    Full-text · Article · May 2007 · Protein Science
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    • "A useful complement to H(CC)H-TOCSY is the HC(CCO)NH (Grzesiek et al. 1993; Mobli et al. 2010; Zuiderweg et al. 1996), where, after the initial 1 H- 13 C INEPT step, 13 C-coherences on the side chains are transferred through TOCSY mixing to the C a -position and through subsequent INEPT steps via the carbonyl to the amide of the following residue (Logan et al. 1992; Montelione et al. 1992). Here, the side chain assignment is facilitated by the connectivity to the well-dispersed backbone amide nuclei 1 H and 15 N. Despite sensitivity losses during the long transfer delays, this approach is especially advantageous for large proteins ([30 kDa) as uniform deuteration combined with specific protonation of methyl groups of interest, as well as the TROSY principle, can be exploited (Gardner et al. 1996; Goto et al. 1999; Hilty et al. 2002; Lin and Wagner 1999; Tugarinov and Kay 2003). The combined information from the two experiments H(CC)H-TOCSY and HC(CCO)NH yields side chain assignments of all aliphatic residues and connects these to the backbone. "
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    ABSTRACT: The assignment of the aliphatic 13C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) 13C-start and 13C-observe NMR experiments. These experiments start with 13C excitation and end with 13C observation while relying on the long transverse relaxation times of 13C spins in uniformly deuterated and 13C,15N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce 13C T 1 relaxation times with little effect on 13C T 2 relaxation times. Such 2D 13C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain 1H spins, the transfer of the side-chain spin polarization to the 1HN spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic 13C resonances, the side-chain 1H chemical shifts can be assigned in a semiautomatic fashion using 3D 15N-resolved and 13C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the 1H,1H NOEs in combination with multiple residual dipolar couplings between adjacent 13C spins determined with 2D 13C-only experiments.
    Full-text · Article · Jul 2003 · Journal of Biomolecular NMR
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