title: Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.
abstract: 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 ~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.
authors.affiliation: †Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Maxim A
authors.initials: MA
authors.lastname: Voinov
authors.forename: Daryl B
authors.initials: DB
authors.lastname: Good
authors.forename: Meaghan E
authors.initials: ME
authors.lastname: Ward
authors.affiliation: †Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Sergey
authors.initials: S
authors.lastname: Milikisiyants
authors.affiliation: †Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Antonin
authors.initials: A
authors.lastname: Marek
authors.affiliation: ∥Bruker Biospin Ltd., Billerica, Massachusetts 01821, United States.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Caporini
authors.affiliation: ∥Bruker Biospin Ltd., Billerica, Massachusetts 01821, United States.
authors.forename: Melanie
authors.initials: M
authors.lastname: Rosay
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Munro
authors.forename: Milena
authors.initials: M
authors.lastname: Ljumovic
authors.forename: Leonid S
authors.initials: LS
authors.lastname: Brown
authors.forename: Vladimir
authors.initials: V
authors.lastname: Ladizhansky
authors.affiliation: †Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Alex I
authors.initials: AI
authors.lastname: Smirnov
mesh_terms: D017033:Anabaena; D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry; D008698:Mesylates / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D009154:Mutation; D009587:Nitrogen Isotopes / Q000737:chemistry; D009589:Nitrogen Oxides / Q000138:chemical synthesis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D020005:Propanols / Q000737:chemistry; D011522:Protons; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000235:genetics; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: C521769:1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol; D003497:Cyclic N-Oxides; D008698:Mesylates; D009587:Nitrogen Isotopes; D009589:Nitrogen Oxides; D020005:Propanols; D011522:Protons; D025603:Sensory Rhodopsins; D012997:Solvents; C000608513:ToSMTSL compound; D014867:Water; C074502:methanethiosulfonate; C039900:nitroxyl; D003545:Cysteine; D005990:Glycerol