Disc antenna enhanced infrared spectroscopy: From felf-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

structure and mechanism of a light-gated cation channel

The new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10− 12 s to 10− 3 s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed

Channelrhodopsin unchained: Structure and mechanism of a light-gated cation channel

AbstractThe new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10−12s to 10−3s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks

Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins

AbstractSurface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane. To this end, the metal surface used in SEIRAS is chemically modified to generate an oriented membrane protein film. Monolayers of uniformly oriented membrane proteins are formed by tethering His-tagged proteins to a nickel nitrilo-triacetic acid (Ni-NTA) modified gold surface and SEIRAS commands molecular sensitivity to probe each step of surface modification. The solid surface used as plasmonic substrate for SEIRAS, can also be employed as an electrode to investigate systems where electron transfer reactions are relevant, like e.g. cytochrome c oxidase or plant-type photosystems. Furthermore, the interaction of these membrane proteins with water-soluble proteins, like cytochrome c or hydrogenase, is studied on the molecular level by SEIRAS. The impact of the membrane potential on protein functionality is verified by monitoring light–dark difference spectra of a monolayer of sensory rhodopsin (SRII) at different applied potentials. It is demonstrated that the interpretations of all of these experiments critically depend on the orientation of the solid-supported membrane protein. Finally, future directions of SEIRAS including cellular systems are discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies

Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s

We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800–1700 cm− 1) and on the CO rebinding reaction of myoglobin (1960–1840 cm− 1), at a spectral resolution of 1 cm− 1. The spectrometric setup covers the time range from 4 ns to nearly a second with a response time of 10–15 ns. Absorption changes as low as 1 × 10− 4 are detected in single-shot experiments at t > 1 μs, and of 5 × 10− 6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H2O)

Evaluating aliphatic CF, CF2, and CF3 groups as vibrational Stark effect reporters

Given the extensive use of fluorination in molecular design, it is imperative to understand the solvation properties of fluorinated compounds and the impact of the C–F bond on electrostatic interactions. Vibrational spectroscopy can provide direct insights into these interactions by using the C–F bond stretching [v(C–F)] as an electric field probe through the vibrational Stark effect (VSE). In this work, we explore the VSE of the three basic patterns of aliphatic fluorination, i.e., mono-, di-, and trifluorination in CF, CF2, and CF3 groups, respectively, and compare their response to the well-studied aromatic v(C–F). Magnitudes (i.e., Stark tuning rates) and orientations of the difference dipole vectors of the v(C–F)-containing normal modes were determined using density functional theory and a molecular dynamics (MD)-assisted solvatochromic analysis of model compounds in solvents of varying polarity. We obtain Stark tuning rates of 0.2–0.8 cm−1/(MV/cm), with smallest and largest electric field sensitivities for CFaliphatic and CF3,aliphatic, respectively. While average electric fields of solvation were oriented along the main symmetry axis of the CFn, and thus along its static dipole, the Stark tuning rate vectors were tilted by up to 87° potentially enabling to map electrostatics in multiple dimensions. We discuss the influence of conformational heterogeneity on spectral shifts and point out the importance of multipolar and/or polarizable MD force fields to describe the electrostatics of fluorinated molecules. The implications of this work are of direct relevance for studies of fluorinated molecules as found in pharmaceuticals, fluorinated peptides, and proteins

How [FeFe]-hydrogenase facilitates bidirectional proton transfer

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases

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