A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15N and 1H components of NH2 multiplets. Although even modest sensitivity gains in 2D NH2-TROSY correlation maps compared to their decoupled NH2â??HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5Â*ÂșC. The NH2-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH2 positions of the L99A T4 lysozyme mutantÂ*â??Â*a model system for studies of the role of protein dynamics in ligand binding.
Impact of two-bond 15 Nâ?? 15 N scalar couplings on 15 N transverse relaxation measurements for arginine side chains of proteins
Impact of two-bond 15 Nâ?? 15 N scalar couplings on 15 N transverse relaxation measurements for arginine side chains of proteins
Abstract
NMR relaxation of arginine (Arg) 15NΔ nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15Nâ??15N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15NΔ nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15Nâ??15N...
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[NMR paper] Selective (15)N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy.
Selective (15)N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy.
Related Articles Selective (15)N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy.
J Biomol NMR. 2014 Jul 8;
Authors: Cao C, Chen JL, Yang Y, Huang F, Otting G, Su XC
Abstract
The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in...
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07-10-2014 08:25 AM
Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
Abstract
The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective 15N-labeling of side-chain amides, however, can be a challenge due to enzyme-catalyzed exchange of amide groups during protein synthesis. In the present study, we developed an efficient way of selectively...
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07-07-2014 11:50 PM
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Abstract
Protein dynamics on the microsecondâ??millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the...
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06-19-2014 10:21 PM
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Abstract Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding...
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07-30-2012 07:42 AM
13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy
13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy
Abstract Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of 13C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of...
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07-05-2012 04:13 AM
[NMR paper] Relaxation-optimized NMR spectroscopy of methylene groups in proteins and nucleic aci
Relaxation-optimized NMR spectroscopy of methylene groups in proteins and nucleic acids.
Related Articles Relaxation-optimized NMR spectroscopy of methylene groups in proteins and nucleic acids.
J Am Chem Soc. 2004 Sep 1;126(34):10560-70
Authors: Miclet E, Williams Jr DC, Clore GM, Bryce DL, Boisbouvier J, Bax A
A large fraction of hydrogens in proteins and nucleic acids is of the methylene type. Their detailed study, however, in terms of structure and dynamics by NMR spectroscopy is hampered by their fast relaxation properties, which give...
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11-24-2010 10:01 PM
[NMR paper] Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX
Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles.
Related Articles Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2358-63
Authors: Fernández C, Adeishvili K, Wüthrich K
The (2)H,(13)C,(15)N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles...
Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins
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