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.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

“…Measurements of the H-C14-C15-H retinal torsional angle revealed a significant out-of-plane twist with an angle of 158°± 2°. A similar out-of-plane twist has also been observed for bacteriorhodopsin (164°) (53) and green proteorhodopsin (161°) (47), indicating that this out-of-plane twist is a general property of microbial retinal rhodopsins and might help to provide a favorable orientation of the Schiff base during the subsequent photocycle steps. (Fig.…”

“…2C). The obtained value of 1.51 ± 0.02 Å is significantly longer than the 1.42 Å observed for green proteorhodopsin (47). This increase in bond length corresponds well with a lower double-bond character of the bond as expected from the blue shift of the absorption maximum compared with green proteorhodopsin.…”

“…This technique requires temperatures around 100 K that are also compatible with trapping of photointermediates as outlined below. DNP-enhanced MAS NMR is not yet a routine method but is applied increasingly to complex, mechanistic studies on retinal proteins (45)(46)(47)(48) and other membrane proteins (49)(50)(51).…”

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

“…Because of the small number of spins and the small amount of labeled retinal available, signal enhancement on the basis of DNP was applied. Using TOTAPOL (27) as a polarizing agent, a 20-fold signal enhancement was achieved, as reported previously (21,22). (Fig.…”

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…A major issue in MAS ssNMR is obtaining high quality spectra (good signal-to-noise ratio, high spectral resolution). The growing repertoire of polarization transfer schemes (reviewed in [68][69][70][71]), access to high magnetic fields (up to 1 GHz), the development of fast to ultra-fast MAS probes [72][73][74] and associated methods [75,76], the development of efficient rotor filling procedures [77,78], the use of specific labeling schemes [79][80][81][82][83], the development of proton detected experiments [74,[84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101], and biomolecular dynamic nuclear polarization [60,[102][103][104][105][106][107][108][109][110][111][112]<...>…”