Voinov, M.A., et al., Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. J Phys Chem B, 2015. 119(32): p. 10180-90.
Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with approximately 17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.
[NMR paper] Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.
Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.
Related Articles Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.
J Phys Chem B. 2015 Jul 31;
Authors: Voinov MA, Good DB, Ward ME, Milikisiyants S, Marek A, Caporini MA, Rosay M, Munro RA, Ljumovic M, Brown LS, Ladizhansky V, Smirnov AI
Abstract
Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from...
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08-02-2015 07:10 AM
Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR
Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR
Jakob Maciejko, Michaela Mehler, Jagdeep Kaur, Tobias Lieblein, Nina Morgner, Olivier Ouari, Paul Tordo, Johanna Becker-Baldus and Clemens Glaubitz
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/jacs.5b03606/20150713/images/medium/ja-2015-03606j_0008.gif
Journal of the American Chemical Society
DOI: 10.1021/jacs.5b03606
...
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07-14-2015 02:59 AM
Static (1)H dynamic nuclear polarization with the biradical TOTAPOL: a transition between the solid effect and the cross effect
From The DNP-NMR Blog:
Static (1)H dynamic nuclear polarization with the biradical TOTAPOL: a transition between the solid effect and the cross effect
Shimon, D., et al., Static (1)H dynamic nuclear polarization with the biradical TOTAPOL: a transition between the solid effect and the cross effect. Phys Chem Chem Phys, 2014. 16(14): p. 6687-99.
http://www.ncbi.nlm.nih.gov/pubmed/24585094
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06-04-2014 03:22 PM
Synthesis and evaluation of nitroxide-based oligoradicals for low-temperature dynamic nuclear polarization in solid state NMR
From The DNP-NMR Blog:
Synthesis and evaluation of nitroxide-based oligoradicals for low-temperature dynamic nuclear polarization in solid state NMR
Yau, W.-M., K.R. Thurber, and R. Tycko, Synthesis and evaluation of nitroxide-based oligoradicals for low-temperature dynamic nuclear polarization in solid state NMR. J. Magn. Reson., 2014(0).
http://www.sciencedirect.com/science/article/pii/S1090780714001372
Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation
From The DNP-NMR Blog:
Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation
This article was already published in 2009 but unfortunately I missed it.
Pavlova, A., et al., Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation. Phys. Chem. Chem. Phys., 2009. 11(31): p. 6833-6839.
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11-21-2013 01:14 AM
Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K
From The DNP-NMR Blog:
Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K
Zagdoun, A., et al., Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K. J. Am. Chem. Soc., 2013. 135(34): p. 12790-12797.
http://dx.doi.org/10.1021/ja405813t
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09-25-2013 11:34 PM
Large Molecular Weight Nitroxide Biradicals Providing Effi-cient Dynamic Nuclear Polarization at Temperatures up to 200 Kelvin
From The DNP-NMR Blog:
Large Molecular Weight Nitroxide Biradicals Providing Effi-cient Dynamic Nuclear Polarization at Temperatures up to 200 Kelvin
Zagdoun, A., et al., Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K. J Am Chem Soc, 2013. 135(34): p. 12790-7.
http://www.ncbi.nlm.nih.gov/pubmed/23961876
Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR
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