A new group of eubacterial light-driven retinal-binding proton pumps with an unusual cytoplasmic proton donor.

One of the main functions of microbial rhodopsins is outward-directed light-driven proton transport across the plasma membrane, which can provide sources of energy alternative to respiration and chlorophyll photosynthesis. Proton-pumping rhodopsins are found in Archaea (Halobacteria), multiple groups of Bacteria, numerous fungi, and some microscopic algae. An overwhelming majority of these proton pumps share the common transport mechanism, in which a proton from the retinal Schiff base is first transferred to the primary proton acceptor (normally an Asp) on the extracellular side of retinal. Next, reprotonation of the Schiff base from the cytoplasmic side is mediated by a carboxylic proton donor (Asp or Glu), which is located on helix C and is usually hydrogen-bonded to Thr or Ser on helix B. The only notable exception from this trend was recently found in Exiguobacterium, where the carboxylic proton donor is replaced by Lys. Here we describe a new group of efficient proteobacterial retinal-binding light-driven proton pumps which lack the carboxylic proton donor on helix C (most often replaced by Gly) but possess a unique His residue on helix B. We characterize the group spectroscopically and propose that this histidine forms a proton-donating complex compensating for the loss of the carboxylic proton donor.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

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.