Related ArticlesGlobal folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
Biochemistry. 1997 Feb 11;36(6):1389-401
Authors: Gardner KH, Rosen MK, Kay LE
The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deuterons significantly improves the sensitivity of many of the experiments used in chemical shift assignment. Unfortunately, uniform deuteration also radically depletes the number of interproton distance restraints available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterated proteins through the use of deuterated, selectively methyl-protonated samples. This labeling profile maintains the efficiency of triple-resonance NMR experiments while retaining a sufficient number of protons at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how this deuteration scheme affects the sensitivity and resolution of experiments used to assign 15N, 13C, and 1H chemical shifts and interproton NOEs. This approach is tested experimentally on a 14 kDa SH2/phosphopeptide complex, and a global protein fold is obtained from a set of methyl-methyl, methyl-NH, and NH-NH distance restraints. We demonstrate that the inclusion of methyl-NH and methyl-methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH-NH distance restraints. Finally, we examine the general applicability of this approach by determining the structures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.
Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy
Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy
Abstract Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743â??1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the build-up of methyl proton multiple-quantum coherences that can be created in magnetically equivalent...
nmrlearner
Journal club
0
02-11-2012 10:31 AM
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
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...
nmrlearner
Journal club
0
09-26-2011 06:42 AM
Radio frequency assisted homonuclear recoupling - A Floquet description of homonuclear recoupling via surrounding heteronuclei in fully protonated to fully deuterated systems
Radio frequency assisted homonuclear recoupling - A Floquet description of homonuclear recoupling via surrounding heteronuclei in fully protonated to fully deuterated systems
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 18 January 2011</br>
Michal, Leskes , Ümit, Akbey , Hartmut, Oschkinat , Barth-Jan, van Rossum , Shimon, Vega</br>
We present a Floquet theory approach for the analysis of homonuclear recoupling assisted by radio frequency (RF) irradiation of surrounding heteronuclear spins. This description covers a...
nmrlearner
Journal club
0
01-19-2011 03:04 PM
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Abstract Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from -glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple â??out-and-backâ?? NMR methodology. Such selective methyl-detected â??out-and-backâ?? NMR experiments allow complete assignments of threonine γ2...
nmrlearner
Journal club
0
01-09-2011 12:46 PM
A simple strategy for 13C,1H labeling at the Ile-γ2 methyl position in highly deuter
A simple strategy for 13C,1H labeling at the Ile-γ2 methyl position in highly deuterated proteins
Abstract A straightforward approach for the production of highly deuterated proteins labeled with 13C and 1H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile 13Cγ2,1Hγ2 HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile 13Cδ1,1Hδ1 spectra. This labeling scheme adds to a growing...
nmrlearner
Journal club
0
10-20-2010 06:50 AM
[NMR paper] Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR
Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
Biochemistry. 1997 Feb 11;36(6):1389-401
Authors: Gardner KH, Rosen MK, Kay LE
The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses...
nmrlearner
Journal club
0
08-22-2010 03:03 PM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
administrator
Solid-state high-res. NMR
1
08-05-2009 03:21 AM
Help!!Why does a deuterated protein behave even more poorly than the protonated one?
A 20kDa protein, about half of the total signals can be observed in CBCA(CO)NH experiment when protonated with sample concentration of 1mM. However, after deuterated, hardly any signals can be observed with the same concentration in this experiment. We use the same pulse sequence except adding deuterium decoupling in the deuterated one. I don't know why? Help!!
Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR
Linear Mode
