Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better... Show moreOpsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize QuasAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable M-like form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed. Show less
Multi-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested... Show moreMulti-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested the efficacy of several commonly used detergents and membrane-mimicking nanodiscs with respect to solubilization, spectral properties, thermal stability and oligomeric profile of two membrane proteins from the eubacterial rhodopsin family, green proteorhodopsin (PR) and Gloeobacter violaceus rhodopsin (GR). Good solubilization was observed for the detergents TritonX-100 and dodecylphosphocholine (DPC), but DPC in particular strongly affected the thermal stability of PR and especially GR. The least deleterious effects were obtained with n-dodecyl-β-D-maltopyranoside (DDM) and octyl glucose neopentyl glycol (OGNG), which adequately stabilized the native oligomeric and monomeric state of PR and GR, respectively. The transition from the oligomeric to the monomeric state is accompanied by a small red-shift. Both GR and PR were rather unstable in SMA-nanodiscs, but the highest thermal stability was realized by the MSP-nanodisc environment. The size of the MSP-nanodisc was too small to fit the PR hexamer, but large enough to contain the PR monomer and GR trimer. This permitted the comparison of the photocycle of trimeric GR in a membrane-mimicking (MSP-nanodisc) and a detergent (DDM) environment. The ultrarapid early phase of the photocycle (femto- to picosecond lifetimes) showed very similar kinetics in either environment, but the slower part, initiated with proton transfer and generation of the M intermediate, proceeded faster in the nanodisc environment. The implications of our results for the biophysical characterization of PR and GR are discussed. Show less
Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals.... Show moreOpsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging. Show less
Multi-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested... Show moreMulti-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested the efficacy of several commonly used detergents and membrane-mimicking nanodiscs with respect to solubilization, spectral properties, thermal stability and oligomeric profile of two membrane proteins from the eubacterial rhodopsin family, green proteorhodopsin (PR) and Gloeobacter violaceus rhodopsin (GR). Good solubilization was observed for the detergents TritonX-100 and dodecylphosphocholine (DPC), but DPC in particular strongly affected the thermal stability of PR and especially GR. The least deleterious effects were obtained with n-dodecyl-β-D-maltopyranoside (DDM) and octyl glucose neopentyl glycol (OGNG), which adequately stabilized the native oligomeric and monomeric state of PR and GR, respectively. The transition from the oligomeric to the monomeric state is accompanied by a small red-shift. Both GR and PR were rather unstable in SMA-nanodiscs, but the highest thermal stability was realized by the MSP-nanodisc environment. The size of the MSP-nanodisc was too small to fit the PR hexamer, but large enough to contain the PR monomer and GR trimer. This permitted the comparison of the photocycle of trimeric GR in a membrane-mimicking (MSP-nanodisc) and a detergent (DDM) environment. The ultrarapid early phase of the photocycle (femto- to picosecond lifetimes) showed very similar kinetics in either environment, but the slower part, initiated with proton transfer and generation of the M intermediate, proceeded faster in the nanodisc environment. The implications of our results for the biophysical characterization of PR and GR are discussed. Show less
Microbial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared... Show moreMicrobial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared spectral regions is of great demand in such applications because more bathochromic light into the near-infrared range penetrates deeper in living tissue. Recently, retinal analogues have been successfully used in ion transporting and fluorescent rhodopsins to achieve red-shifted absorption, activity, and emission properties. Understanding their photochemical mechanism is essential for further design of appropriate retinal analogues but is yet only poorly understood for most retinal analogue pigments. Here, we report the photoreaction dynamics of red-shifted analogue pigments of the proton pump proteorhodopsin (PR) containing A2 (all-trans-3,4-dehydroretinal), MOA2 (all-trans-3-methoxy-3,4-dehydroretinal), or DMAR (all-trans-3-dimethylamino-16-nor-1,2,3,4-didehydroretinal), utilizing femto- to submillisecond transient absorption spectroscopy. We found that the A2 analogue photoisomerizes in 1.4, 3.0, and/or 13 ps upon 510 nm light illumination, which is comparable to the native retinal (A1) in PR. On the other hand, the deprotonation of the A2 pigment Schiff base was observed with a dominant time constant of 67 μs, which is significantly slower than the A1 pigment. In the MOA2 pigment, no isomerization or photoproduct formation was detected upon 520 nm excitation, implying that all the excited molecules returned to the initial ground state in 2.0 and 4.2 ps. The DMAR pigment showed very slow excited state dynamics similar to the previously studied MMAR pigment, but only very little photoproduct was formed. The low efficiency of the photoproduct formation likely is the reason why DMAR analogue pigments of PR showed very weak proton pumping activity. Show less
Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and... Show moreMicrobial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore. Show less
19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local... Show more19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local environment. In this study we have exploited a fluorine substituent, strategically placed at the C-12-position of 11-cis retinal, the chromophore of visual rhodopsins. This label was used to explore the local environment of the chromophore in the ground state of bovine rhodopsin and its active photo-intermediate Meta II. In addition, the chemical shift and tensor elements of the chromophore in the free state in a membrane environment and the bound state in the protein were determined. Upon binding of the chromophore into rhodopsin and Meta II, the isotropic chemical shift changes in the opposite direction by +9.7 and -8.4 ppm, respectively. An unusually large isotropic shift difference of 35.9 ppm was observed between rhodopsin and Meta II. This partly originates in the light-triggered 11-cis to all-trans isomerization of the chromophore. The other part reflects the local conformational rearrangements in the chromophore and the binding pocket. These NMR data were correlated with the available X-ray structures of rhodopsin and Meta II using bond polarization theory. For this purpose hydrogen atoms have to be inserted and hereto a family of structures were derived that best correlated with the well-established 13C chemical shifts. Based upon these structures, a 12-F derivative was obtained that best corresponded with the experimentally determined 19F chemical shifts and tensor elements. The combined data indicate strong changes in the local environment of the C-12 position and a substantially different interaction pattern with the protein in Meta II as compared to rhodopsin. Show less
(encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light... Show more(encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis. Show less
Buda, F.; Keijer, T.; Ganapathy, S.; Grip, W.J. de 2017
Proteorhodopsin is a light‐driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all‐trans retinal A1 chromophore covalently bound to a lysine residue via a protonated... Show moreProteorhodopsin is a light‐driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all‐trans retinal A1 chromophore covalently bound to a lysine residue via a protonated Schiff base. In this study, we exploited density functional theory (DFT) calculations to investigate the retinal binding pocket in the dark state and after mimicking photoisomerization. The model of the binding pocket is constructed incrementally by adding the residues near the retinal that are necessary to ensure a stable protonated Schiff base. The presence of a few water molecules near the Schiff base turns out to be an essential feature of the model. The absorption properties are then studied using time‐dependent DFT (TDDFT) and compared to experimental data to further validate the structural model and to assess the accuracy of the computational setting. It is shown that TDDFT is able to reproduce the main absorption peak accurately and to quantitatively determine the spectral shift induced by substituting the native all‐trans retinal A1 chromophore with different retinal analogues. Moreover, ab initio molecular dynamics simulations are performed to investigate the vibrational spectra of our models before and after isomerization. Specific differences in the vibrational spectra are identified that provide further insight into experimental FTIR difference spectra. Show less
A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively... Show moreA recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet–visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C=O stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 → Gln and Ser97 → Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L Show less
