Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells.

The ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger.

Proton Release Reactions in the Inward H+ Pump NsXeR.

Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.

Assessing the Role of R120 in the Gating of CrChR2 by Time-Resolved Spectroscopy from Femtoseconds to Seconds.

The light-gated ion channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of CrChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in CrChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.

The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy.

Cytochrome c oxidase (CcO) is a transmembrane heme­copper metalloenzyme that catalyzes the reduction of O2 to H2O at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited R2CO state as well as the fully-reduced R4 and R4CO states and induced the "active ready" oxidized state OH. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O2, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a3 does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R2CO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a3 in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO.

Time-resolved photoacoustics of channelrhodopsins: early energetics and light-driven volume changes.

In biological photoreceptors, the energy stored in early transient species is a key feature to drive the photocycle or a chain of reactions. Time-resolved photoacoustics (PA) can explore the energy landscape of transient species formed within few ns after photoexcitation, as well as volumetric changes (ΔV) of these intermediates with respect to the parental state. In this work, PA identified these important parameters for several channelrhodopsins, namely CaChR1 from Chlamydomonas augustae and CrChR2 from Chlamydomonas reinhardtii and various variants. PA has access to the sub-ns formation of the early photoproduct P1 and to its relaxation, provided that this latter process occurs within a few µs. We found that ΔVP1 for CaChR1 is ca. 12 mL/mol, while it is much smaller for CrChR2 (4.7 mL/mol) and for H. salinarum bacteriorhodopsin (HsBR, ΔVK = 2.8 mL/mol). PA experiments on variants strongly indicate that part of this large ΔVP1 value for CaChR1 is caused by the protonation dynamics of the Schiff base counterion complex involving E169 and D299. PA data further show that the energy level of P1 is higher in CrChR2 (ca. 96 kJ/mol) than in CaChr1 (ca. 46 kJ/mol), comparable to the energy level of the K state of HsBR (60 kJ/mol). Instrumental to gain these molecular values from the raw PA data was the estimation of the quantum yield (Φ) for P1 formation via transient spectroscopy; for both channelrhodopsins, ΦP2 was evaluated as ca. 0.4.

Monitoring the Progression of Cell-Free Expression of Microbial Rhodopsins by Surface Enhanced IR Spectroscopy: Resolving a Branch Point for Successful/Unsuccessful Folding.

The translocon-unassisted folding process of transmembrane domains of the microbial rhodopsins sensory rhodopsin I (HsSRI) and II (HsSRII), channelrhodopsin II (CrChR2), and bacteriorhodopsin (HsBR) during cell-free expression has been investigated by Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS). Up to now, only a limited number of rhodopsins have been expressed and folded into the functional holoprotein in cell free expression systems, while other microbial rhodopsins fail to properly bind the chromophore all-trans retinal as indicated by the missing visible absorption. SEIRAS experiments suggest that all investigated rhodopsins lead to the production of polypeptides, which are co-translationally inserted into a solid-supported lipid bilayer during the first hour after the in-vitro expression is initiated. Secondary structure analysis of the IR spectra revealed that the polypeptides form a comparable amount of α-helical structure during the initial phase of insertion into the lipid bilayer. As the process progressed (>1 h), only HsBR exhibited a further increase and association of α-helices to form a compact tertiary structure, while the helical contents of the other rhodopsins stagnated. This result suggests that the molecular reason for the unsuccessful cell-free expression of the two sensory rhodopsins and of CrChR2 is not due to the translation process, but rather to the folding process during the post-translational period. Taking our previous observation into account that HsBR fails to form a tertiary structure in the absence of its retinal, we infer that the chromophore retinal is an integral component of the compaction of the polypeptide into its tertiary structure and the formation of a fully functional protein.

Photoactivation of a Mechanosensitive Channel.

Optogenetics in the conventional sense, i.e. the use of engineered proteins that gain their light sensitivity from naturally abundant chromophores, represents an exciting means to trigger and control biological activity by light. As an alternate approach, photopharmacology controls biological activity with the help of synthetic photoswitches. Here, we used an azobenzene-derived lipid analogue to optically activate the transmembrane mechanosensitive channel MscL which responds to changes in the lateral pressure of the lipid bilayer. In this work, MscL has been reconstituted in nanodiscs, which provide a native-like environment to the protein and a physical constraint to membrane expansion. We characterized this photomechanical system by FTIR spectroscopy and assigned the vibrational bands of the light-induced FTIR difference spectra of the trans and cis states of the azobenzene photolipid by DFT calculations. Differences in the amide I range indicated reversible conformational changes in MscL as a direct consequence of light switching. With the mediation of nanodiscs, we inserted the transmembrane protein in a free standing photoswitchable lipid bilayer, where electrophysiological recordings confirmed that the ion channel could be set to one of its sub-conducting states upon light illumination. In conclusion, a novel approach is presented to photoactivate and control cellular processes as complex and intricate as gravitropism and turgor sensing in plants, contractility of the heart, as well as sensing pain, hearing, and touch in animals.

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy.

It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids' collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.

The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis.

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

Nanodisc Reconstitution of Channelrhodopsins Heterologously Expressed in Pichia pastoris for Biophysical Investigations.

For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.

Near-Infrared Activation of Sensory Rhodopsin II Mediated by NIR-to-Blue Upconversion Nanoparticles.

Direct optical activation of microbial rhodopsins in deep biological tissue suffers from ineffective light delivery because visible light is strongly scattered and absorbed. NIR light has deeper tissue penetration, but NIR-activation requires a transducer that converts NIR light into visible light in proximity to proteins of interest. Lanthanide-doped upconversion nanoparticles (UCNPs) are ideal transducer as they absorb near-infrared (NIR) light and emit visible light. Therefore, UCNP-assisted excitation of microbial rhodopsins with NIR light has been intensively studied by electrophysiology technique. While electrophysiology is a powerful method to test the functional performance of microbial rhodopsins, conformational changes associated with the NIR light illumination in the presence of UCNPs remain poorly understood. Since UCNPs have generally multiple emission peaks at different wavelengths, it is important to reveal if UCNP-generated visible light induces similar structural changes of microbial rhodopsins as conventional visible light illumination does. Here, we synthesize the lanthanide-doped UCNPs that convert NIR light to blue light. Using these NIR-to-blue UCNPs, we monitor the NIR-triggered conformational changes in sensory rhodopsin II from Natronomonas pharaonis (NpSRII), blue light-sensitive microbial rhodospsin, by FTIR spectroscopy. FTIR difference spectrum of NpSRII was recorded under two different excitation conditions: (ⅰ) with conventional blue light, (ⅱ) with UCNP-generated blue light upon NIR excitation. Both spectra display similar spectral features characteristic of the long-lived M photointermediate state during the photocycle of NpSRII. This study demonstrates that NIR-activation of NpSRII mediated by UCNPs takes place in a similar way to direct blue light activation of NpSRII.

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase.

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Author Correction: Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase.

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.

Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump-IR-Probe Spectroscopy and Computational Methods.

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivity.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.

Photoexcitation of the P4480 State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2.

Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P3520 intermediate of the primary all- trans photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P4480, the last intermediate of the all- trans photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I3530. Our results indicate that the D2 and O2 states correspond to the P4480 and I3530 intermediates, connecting desensitization of ChR2 with the photochemical properties of the P4480 intermediate.

Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins.

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 104 giving rise to a detection limit <1 femtomol (10-15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region.