Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins.

Three all-trans retinals containing multiple 13 C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsin and channelrhodopsin. The synthetic approaches allowed specific introduction of 13 C labels in ring substituents and at different positions in the polyene chain to probe structural features such as ring orientation and interaction of the chromophore with the protein in the ground state and in photointermediates. [10-18-13 C9 ]-All-trans-retinal (1b), [12,15-13 C2 ]-all-trans-retinal (1c), and [14,15-13 C2 ]-all-trans-retinal (1d) were synthesized in in 12, 8, and 7 linear steps from ethyl 2-oxocyclohexanecarboxylate (5) or ß-ionone (4), respectively.

Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR.

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the deprotonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.

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

Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR.

Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.

Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy.

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.

The EF loop in green proteorhodopsin affects conformation and photocycle dynamics.

The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced (13)C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.