Vibrational Study of the Inward Proton Pump Xenorhodopsin NsXeR: Switch Order Determines Vectoriality.

Common proton pumps, e.g. HsBR and PR, transport protons out of the cell. Xenorhodopsins (XeR) were the first discovered microbial rhodopsins which come as natural inward proton pumps. In this work we combine steady-state (cryo-)FTIR and Raman spectroscopy with time-resolved IR and UV/Vis measurements to roadmap the inward proton transport of NsXeR and pinpoint the most important mechanistic features. Through the assignment of characteristic bands of the protein backbone, the retinal chromophore, the retinal Schiff base and D220, we could follow the switching processes for proton accessibility in accordance with the isomerization / switch / transfer model. The corresponding transient IR signatures suggest that the initial assignment of D220 as the proton acceptor needs to be questioned due to the temporal mismatch of the Schiff base and D220 protonation steps. The switching events in the K-L and MCP-MEC transitions are finely tuned by changes of the protein backbone and rearrangements of the Schiff base. This finely tuned mechanism is disrupted at cryogenic temperatures, being reflected in the replacement of the previously reported long-lived intermediate GS* by an actual redshifted (O-like) intermediate.

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.

Photochemistry of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2 and Its Implications on Microbial Rhodopsin Research: Retrospective and Perspective.

The discovery of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) in 2013 has changed the paradigm that cation transport in microbial rhodopsins is restricted to the translocation of protons. Even though this finding is already remarkable by itself, it also reignited more general discussions about the functional mechanism of ion transport. The unique composition of the retinal binding pocket in KR2 with a tight interaction between the retinal Schiff base and its respective counterion D116 also has interesting implications on the photochemical pathway of the chromophore. Here, we discuss the most recent advances in our understanding of the KR2 functionality from the primary event of photon absorption by all-trans retinal up to the actual protein response in the later phases of the photocycle, mainly from the point of view of optical spectroscopy. In this context, we furthermore highlight some of the ongoing debates on the photochemistry of microbial rhodopsins and give some perspectives for promising future directions in this field of research.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

Inactivation of Competitive Decay Channels Leads to Enhanced Coumarin Photochemistry.

In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin- based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen-bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent-dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.

Ultrafast and efficient energy transfer in a one- and two-photon sensitized rhodamine-BODIPY dyad: a perspective for broadly absorbing photocages.

In view of the demand for photoactivatable probes that operate in the visible (VIS) to near infrared (NIR) region of the spectrum, we designed a bichromophoric system based on a rhodamine fluorophore and a BODIPY photocage. Two-photon excited fluorescence (TPEF) measurements and quantum chemical calculations reveal excellent two-photon properties of the employed rhodamine derivative. Excitation of the rhodamine unit via a one- or two-photon process leads to excitation energy transfer (EET) onto the BODIPY part, which is followed by the liberation of the leaving group. Ultrafast transient absorption spectroscopy provides evidence for a highly efficient EET dynamics on a sub-500 femtosecond scale. Complementary quantum dynamical calculations using the multi-layer multiconfiguration time-dependent Hartree (ML-MCTDH) approach highlight the quantum coherent character of the EET transfer. Photorelease of p-nitroaniline (PNA) was investigated by UV/vis absorption spectroscopy by either excitation of the rhodamine or the BODIPY moiety. Even though a quantitative assessment of the PNA yield could not be achieved for this particular BODIPY cage, the present study provides a design principle for a class of photocages that can be broadly activated between 500 and 900 nm.

Transient Near-UV Absorption of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2: A Spectroscopic Marker for Retinal Configuration.

We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S0 → S3 and/or an S0 → S5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR.

The functional mechanism of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13-cis retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures.

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.