title: Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR.
abstract: Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90. Multidimensional spectroscopy of U-(13)C,(15)N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Vikram S
authors.initials: VS
authors.lastname: Bajaj
authors.forename: Melody L
authors.initials: ML
authors.lastname: Mak-Jurkauskas
authors.forename: Marina
authors.initials: M
authors.lastname: Belenky
authors.forename: Judith
authors.initials: J
authors.lastname: Herzfeld
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D008027:Light; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012172:Retinaldehyde / Q000378:metabolism; D013696:Temperature
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D001436:Bacteriorhodopsins; D012172:Retinaldehyde