title: Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy.
abstract: Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and a slight alteration of the retinal chain's out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S0-S1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump-probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.
authors.affiliation: Institute of Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt , 60438 Frankfurt am Main, Germany.
authors.forename: Jiafei
authors.initials: J
authors.lastname: Mao
authors.forename: Nhu-Nguyen
authors.initials: NN
authors.lastname: Do
authors.forename: Frank
authors.initials: F
authors.lastname: Scholz
authors.forename: Lenica
authors.initials: L
authors.lastname: Reggie
authors.forename: Michaela
authors.initials: M
authors.lastname: Mehler
authors.forename: Andrea
authors.initials: A
authors.lastname: Lakatos
authors.forename: Yean-Sin
authors.initials: YS
authors.lastname: Ong
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Ullrich
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Brown
authors.forename: Richard C D
authors.initials: RC
authors.lastname: Brown
authors.forename: Johanna
authors.initials: J
authors.lastname: Becker-Baldus
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D000595:Amino Acid Sequence; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D025604:Rhodopsins, Microbial; C413548:proteorhodopsin