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Dynamicnuclear polarization (DNP) enhances the signal in solid-stateNMR of proteins by transferring polarization from electronic spinsto the nuclear spins of interest. Typically, both the protein andan exogenous source of electronic spins, such as a biradical, areeither codissolved or suspended and then frozen in a glycerol/waterglassy matrix to achieve a homogeneous distribution. While the useof such a matrix protects the protein upon freezing, it also reducesthe available sample volume (by ca. a factor of 4 in our experiments)and causes proportional NMR signal loss. Here we demonstrate an alternativeapproach that does not rely on dispersing the DNP agent in a glassymatrix. We synthesize a new biradical, ToSMTSL, which is based onthe known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonategroup to allow for incorporating this biradical into a protein ina site-directed manner. ToSMTSL was characterized by EPR and testedfor DNP of a heptahelical transmembrane protein, Anabaenasensory rhodopsin (ASR), by covalent modification of solvent-exposedcysteine residues in two 15N-labeled ASR mutants. DNP enhancementswere measured at 400 MHz/263 GHz NMR/EPR frequencies for a seriesof samples prepared in deuterated and protonated buffers and withvaried biradical/protein ratios. While the maximum DNP enhancementof 15 obtained in these samples is comparable to that observed foran ASR sample cosuspended with ∼17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, theachievable sensitivity would be 4-fold greater due to the gain inthe filling factor. We anticipate that the DNP enhancements couldbe further improved by optimizing the biradical structure. The useof covalently attached biradicals would broaden the applicabilityof DNP NMR to structural studies of proteins. [ABSTRACT FROM AUTHOR]