Difference Spectroscopy Research Articles

In vivo detection of changes in cutaneous carotenoids after chemotherapy using shifted excitation resonance Raman difference and fluorescence spectroscopy.

Various cutaneous toxicities under chemotherapy indicate a local effect of chemotherapy by secretion after systemic application. Here, changes in the fluorescence and Raman spectral properties of the stratum corneum subsequent to intravenous chemotherapy were assessed. Twenty healthy subjects and 20 cancer patients undergoing chemotherapy were included. Measurement time points in cancer patients were before the first cycle of chemotherapy (Tbase ) and immediately after intravenous application of the chemotherapy (T1 ). Healthy subjects were measured once without any further intervention. Measurements were conducted using an individually manufactured system consisting of a handheld probe and a wavelength-tunable diode laser-based 488nm SHG light source. Hereby, changes in both skin fluorescence and shifted excitation resonance Raman difference spectroscopy (SERRDS) carotenoid signals were assessed. Healthy subjects showed significantly (P<.001) higher mean concentrations of carotenoids compared to cancer subjects at Tbase . An increase in fluorescence intensity was detected in almost all patients after chemotherapy, especially after doxorubicin infusion. Furthermore, a decrease in the carotenoid concentration in the skin after chemotherapy was found. The SERRDS based noninvasive detection can be used as an indirect quantitative assessment of fluorescent chemotherapeutics. The lower carotenoid SERRDS intensities at Tbase might be due to cancerous diseases and co-medication.

In situ XRD measurements to explore phase formation in the near surface region

Though X-ray diffraction (XRD) is a well-established technique—yet still continuously evolving—for characterization of thin films and diffusion layers, there are two inherent limitations due to the underlying physical principles leading to the diffraction: (i) all data are a weighted integral across the information depth and (ii) X-ray amorphous or nanocrystalline phases are very difficult to be detected. The purpose of this Perspective is to show how a straightforward combination of in situ XRD with depth profiling using a laboratory experiment in combination with a low-energy broad-beam ion source may allow us to overcome these limitations for functional surfaces. Whereas time-resolved investigations of diffusion and phase formation during ion implantation using reactive species are well established, using ion beam sputtering with nonreactive species for depth profiling of the phase composition during the measurements is a rather recent development. While employing analysis of differential spectra leads to a depth resolution below the information depth, ongoing in situ sputtering will lead to information obtained from layers deeper than the information depth. Keeping track of the total diffraction intensity permits the identification of phases without reflections in the XRD measurements, i.e., they appear to be amorphous, and their depth distribution. Selected examples drawn mainly from investigations of the nitriding kinetics of austenitic stainless steel will be used to demonstrate the potential and possible limitations of this method.

De novo resonance assignment of the transmembrane domain of LR11/SorLA in E. coli membranes.

Membrane proteins perform many important cellular functions. Historically, structural studies of these proteins have been conducted in detergent preparations and synthetic lipid bilayers. More recently, magic-angle-spinning (MAS) solid-state NMR has been employed to analyze membrane proteins in native membrane environments, but resonance assignments with this technique remain challenging due to limited spectral resolution and high resonance degeneracy. To tackle this issue, we combine reverse labeling of amino acids, frequency-selective dipolar dephasing, and NMR difference spectroscopy. These methods have resulted in nearly complete resonance assignments of the transmembrane domain of human LR11 (SorLA) protein in E. coli membranes. To reduce background signals from E. coli lipids and proteins and improve spectral sensitivity, we effectively utilize amylose affinity chromatography to prepare membrane vesicles when MBP is included as a fusion partner in the expression construct.

Fourier transform infrared and mass spectrometry analyses of a site-directed mutant of D1-Asp170 as a ligand to the water-oxidizing Mn4CaO5 cluster in photosystem II

The Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), is coordinated by six carboxylate and one imidazole ligands. The roles of these ligands in the water oxidation mechanism remain largely unknown. In this study, we constructed a D1-D170H mutant, in which the Asp ligand bridging Mn and Ca ions was replaced with His, in the cyanobacterium Synechocystis sp. PCC 6803, and analyzed isolated PSII core complexes using Fourier transform infrared (FTIR) difference spectroscopy and mass spectrometry (MS). The S2-minus-S1 FTIR difference spectrum of the PSII complexes of the D1-D170H mutant showed features virtually identical to those of the wild-type PSII. MS analysis further showed that ~70% of D1 proteins from the PSII complexes of D1-D170H possessed the wild-type amino acid sequence, although only the mutated sequence was detected in genomic DNA in the same batch of cells for PSII preparations. In contrast, a D1-S169A mutant as a control showed a modified FTIR spectrum and only a mutated D1 protein. It is thus concluded that the FTIR spectrum of the D1-D170H mutant actually reflects that of wild-type PSII, whereas the Mn4CaO5 cluster is not formed in PSII with D1-D170H mutation. Although the mechanism of production of the wild-type D1 protein in the D1-D170H mutant is unknown at present, a caution is necessary in the analysis of site-directed mutants of crucial residues in the D1 protein, and mutation has to be confirmed not only at the DNA level but also at the amino acid level.

Effect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase

We investigate how isotopic labeling of the enzyme lactate dehydrogenase (LDH) affects its function. LDH is of special interest because there exists a line of residues spanning the protein that are involved in the transition state (TS) of the chemical reaction coordinate (so-called promoting vibration). Hence, studies have been carried out on this protein (as well as others) using labeled protein (so-called heavy protein) along with measurements of single turnover kcat yielding a KIE (=kcatlight/kcatheavy) aimed at understanding the effect of labeling generally and more specifically this line of residues. Here, it is shown that 13C, 15N, and 2H atom labeling of hhLDH (human heart) affects its internal structure which in turn affects its dynamics and catalytic mechanism. Spectral studies employing advanced FTIR difference spectroscopy show that the height of the electronic potential surface of the TS is lowered (probably by ground state destabilization) by labeling. Moreover, laser-induced T-jump relaxation kinetic spectroscopy shows that the microsecond to millisecond nuclear motions internal to the protein are affected by labeling. While the effects are small, they are sufficient to contribute to the observed KIE values as well or even more than promoting vibration effects.

Hydrophobic Hydration of Fluoroalkyl (C–F) is Distinctly Different from That of Its Hydrogenated Counterpart (C–H), as Observed by Raman Difference with Simultaneous Curve Fitting Analysis

Interaction of fluoroalkyl with water is the key parameter tailoring the applications of organofluorines in diverse fields. Nevertheless, how fluorination modifies an alkyl group’s interaction with water and the associated local hydrophobicity is largely unknown. Using Raman difference spectroscopy with simultaneous curve fitting (RD-SCF) and MD simulation, we show that fluorination breaks down the tetrahedral water structure that is otherwise present around the alkyl group (hydrophobic hydration). Electrostatic perturbation (due to fluorination) restricts the water molecules to adopt the symmetrically hydrogen-bonded tetrahedral structure. As a result, the relative population of strongly H-bonded water decreases, while that of the weakly interacting dangling OH increases in the hydration shell of a fluoroalkyl group. These structural changes of water make the accommodation of fluoroalkyl energetically costly compared to that of its hydrogenated counterpart, which is manifested as enhanced hydrophobicity ...

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.

Facile Background Discrimination in Femtosecond Stimulated Raman Spectroscopy Using a Dual-Frequency Raman Pump Technique.

Femtosecondstimulated Raman spectroscopy (FSRS) is a useful technique for uncovering chemical reaction dynamics by acquiring high-resolution Raman spectra with ultrafast time resolution. However, in FSRS, it can be challenging to discern Raman features from signals arising from transient absorption and other four-wave mixing pathways. To overcome this difficulty, we combine the principles of shifted excitation Raman difference spectroscopy with a simple fixed frequency comb to perform dual-frequency Raman pump FSRS. Through the addition of only two mirrors and a slit to the standard FSRS setup, this method provides Raman spectra at two different excitation wavelengths that can be processed by an automated algorithm to reconstruct the Raman spectrum. Here, we demonstrate the utility of dual-frequency Raman pump FSRS to easily identify Raman signatures by visual inspection for excited-state and ground-state spectra, both on- and off-resonance. We show that previously assigned short-lived vibrations of photoexcited β-carotene are actually not vibrational in nature. We also use crystalline betaine-30 as a challenging test case for this method, as the FSRS spectra contain a number of narrow transient vibronic and non-SRS features. By reliably reducing interference from background signals, the interpretation is substantially more quantitative and enables the future study of new systems, particularly those with small Raman cross-sections or solid-state samples with narrow vibronic features.

Quantitation of active pharmaceutical ingredient through the packaging using Raman handheld spectrophotometers: A comparison study

Handheld Raman spectroscopy is actually booming. Recent devices improvements aim at addressing the usual Raman spectroscopy issues: fluorescence with shifted‐excitation Raman difference spectroscopy (SERDS), poor sensitivity with surface enhanced Raman scattering (SERS) and information only about the sample surface with spatially offset Raman spectroscopy (SORS). While qualitative performances of handheld devices are generally well established, the quantitative analysis of pharmaceutical samples remains challenging.The aim of this study was to compare the quantitative performances of three commercially available handheld Raman spectroscopy devices. Two of them (TruScan and IDRaman mini) are equipped with a 785 nm laser wavelength and operate in a conventional backscattering mode. The IDRaman has the Orbital Raster Scanning (ORS) option to increase the analyzed surface. The third device (Resolve) operates with an 830 nm laser wavelength both in backscattering and in SORS modes.The comparative study was carried out on ibuprofen-mannitol-microcrystalline cellulose ternary mixtures. The concentration of ibuprofen ranged from 24 to 52% (w/w) while the proportions of the two excipients were varied to avoid cross-correlation as much as possible. Analyses were performed either directly through a glass vial or with the glass vial in an opaque polypropylene flask, using a validated FT-NIR spectroscopy method as a reference method. Chemometric analyses were carried out with the Partial Least Squares Regression (PLS-R) algorithm. The quantitative models were validated using the total error approach and the ICH Q2 (R1) guidelines with ± 15% as acceptance limits.

Parallelized shifted-excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system.

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.

Shifted Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device (CCD) Lock-In Detection.

Shifted excitation Raman difference spectroscopy (SERDS) can provide effective, chemically specific information on fluorescent samples. However, the restricted ability for fast alternating detection (usually < 10 Hz) of spectra excited at two shifted laser wavelengths can limit its effectiveness when rapidly varying emission backgrounds are present. This paper presents a novel charge-shifting lock-in approach permitting fast SERDS operation (exemplarily demonstrated at 1000 Hz) using a specialized dual-wavelength diode laser (emitting at 829.40 nm and 828.85 nm) and a custom-built charge-coupled device (CCD) enabling charge retention and shifting back and forth on the CCD chip. For six selected mineral samples (moved irregularly during spectral acquisition), results demonstrate superior reproducibility of the fast charge-shifting read-out over the conventional read-out (operated at 5.4 Hz). Partial least squares discriminant analysis revealed improved classification performance of charge-shifting (four latent variables, sensitivity: 99%, specificity: 94%) versus conventional read-out (six latent variables, sensitivity: 90%, specificity: 92%). The charge-shifting concept was also successfully translated to sub-surface analysis using spatially offset Raman spectroscopy (SORS). Charge-shifting SERDS-SORS spectra recorded from a polytetrafluoroethylene layer, concealed behind a 0.25 mm thick, opaque, heterogeneous layer, matched reference spectra much more closely and exhibited a signal-to-background-noise (S/NB) ratio two times higher than that achieved with conventional CCD read-out SERDS-SORS. The novel approach overcomes fundamental limitations of conventional CCDs. In conjunction with the inherent capability of the charge-shifting lock-in technique to suppress rapidly varying ambient light interference demonstrated by us earlier it is expected to be particularly beneficial with heterogeneous fluorescent samples in field applications.

Compact Diode Laser-Based Dual-Wavelength Master Oscillator Power Amplifier at 785 nm

A compact, micro integrated, diode laser-based dual-wavelength master oscillator power amplifier at 785 nm is presented. Laser emission from a Y-branch distributed Bragg reflector laser is coupled into a tilted ridge waveguide amplifier on a $5\times 25$ mm2 micro optical bench. At 25°C and 20 mW pump power, an optical output power of 0.5 W is obtained. The corresponding emission wavelengths are 784.62 and 785.24 nm, resulting in a spectral distance of 10 cm $^{-1}$ . The measured spectral bandwidths and side mode suppression ratios are 0.02 nm (0.3 cm $^{-1}$ ) and 30 dB, respectively. At both wavelengths beam propagation parameters of 1.3 ( $\text{M}{^{2}}_{4\boldsymbol \sigma }$ ) are obtained. This allows for efficient dual-wavelength single-mode fiber coupling. In addition, the device does not show any effects from optical feedback. It neither shows a lateral spatial tilt of the far field intensity distributions for the two wavelengths, previously reported for single Y-branch diode lasers. Based on these parameters, the device is suitable for demanding applications such as shifted excitation Raman difference spectroscopy or confocal Raman microscopy.

Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy.

Two-dimensional nanomembranes are promising materials for filtration or separation by providing the basis for controlled and rapid transport between two compartments. The polymerization by UV light of diacetylene-containing lipids at an interface produces free-standing 2D nanomembranes. Here, we analyzed in situ the nanomembrane formation of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1-palmitoyl-2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE) on germanium using light-induced infrared difference spectroscopy with attenuated total reflection to obtain insights into the kinetics and mechanism of the polymerization process. Our interpretation is supported by atomic force microscopy and density functional theory. Formation of the polymer network is evidenced by changes in the frequency of C═O stretches acting as infrared probes. However, spectral and kinetic analysis revealed a biphasic process in the monolayer. In both phases, losses in signal of CH2 stretches are observed which are not in agreement with the accepted mechanism of chain propagation for diacetylene polymerization. These signals are dominant in the second phase and are assigned to termination reactions with some contributions from intramolecular consecutive reactions. This finding now provides a spectroscopic measure for the identity and integrity of the nanomembrane complementary to microscopic analysis. We deduce that limited 2D mobility on the solid support promotes intramolecular termination, leading to smaller domains.

Deep Learning-Assisted Three-Dimensional Fluorescence Difference Spectroscopy for Identification and Semiquantification of Illicit Drugs in Biofluids.

The fast identification and quantification of illicit drugs in biofluids are of great significance in clinical detection. However, existing drug detection strategies cannot fully meet clinical needs, and the on-site identification and quantification of various illicit drugs in biofluids remain a great challenge. Here, we report the development of a deep learning-assisted three-dimensional (3D) fluorescence difference spectroscopy for rapid identification and semiquantification of illicit drugs in biofluids. This strategy introduces highly fluorescent silver nanoclusters into the biofluids with illicit drugs as signal sources. The interaction between silver nanoclusters and drug molecules changed the fluorescence performance of the mixture. Deep learning methods were applied to grasp the subtle fingerprint information from the 3D fluorescence difference spectra to identify and semiquantify various illicit drugs in biofluids, including codeine, 4,5-methylene-dioxy amphetamine, 3,4-methylene dioxy methamphetamine, meperidine, and methcathinone. This approach can achieve a high prediction accuracy rate of 88.07% and a broad detection range from 2 μg/mL to 100 mg/mL. It opens up a new way for the detection of small molecules with or without fluorescence in complicated matrixes.

Sorption of Water Vapor in Poly(L-Lactic Acid): A Time-Resolved FTIR Spectroscopy Investigation.

In this contribution the sorption of water vapor in Poly(L-lactic acid) (PLLA) was studied by time-resolved FTIR spectroscopy. The collected FTIR data were analyzed by complementary approaches such as difference spectroscopy, two-dimensional correlation spectroscopy (2D-COS), and least-squares curve-fitting analysis which provided information about the overall diffusivity, the nature of the molecular interactions among the polymer and the penetrant and the dynamics of the various molecular species. The diffusion coefficient were evaluated as a function of vapor activity and were found in good agreement with previously reported values. The system showed a Fickian behavior with diffusivity increasing with penetrant concentration. Two distinct water species (first-shell and second-shell layers) were detected and quantified by coupling FTIR and gravimetric measurements.

Fourier transform visible and infrared difference spectroscopy for the study of P700 in photosystem I from Fischerella thermalis PCC 7521 cells grown under white light and far-red light: Evidence that the A–1 cofactor is chlorophyll f

(P700+ – P700) Fourier transform visible and infrared difference spectra (DS) have been obtained using photosystem I (PSI) complexes isolated from cells of Fischerella thermalis PCC 7521 grown under white light (WL) or far-red light (FRL). PSI from cells grown under FRL (FRL-PSI) contain ~8 chlorophyll f (Chl f) molecules (Shen et al., Photosynth. Res. Jan. 2019). Both the visible and infrared DS indicate that neither the PA or PB pigments of P700 are Chl f molecules, but do support the conclusion that at least one of the A–1 cofactors is a Chl f molecule.The FTIR DS indicate that the hydrogen bond to the 131-keto CO group of the PA pigment of P700 is weakened in FRL-PSI, as might be expected given that the proteins that bind the P700 pigments are substantially different in FRL-PSI (Gan et al., Science 345, 1312–1317, 2014).The FTIR DS obtained using FRL-PSI display a band at 1664 cm−1 that is assigned (based on density functional theory calculations) to the 21-formyl CO group of Chl f, that upshifts 5 cm−1 upon P700+ formation. This is much less than expected for a cation-induced upshift, indicating that the Chl f molecule is not one of the pigments of P700.In WL-PSI the A–1 cofactor is a Chl a molecule with 131-keto and 133-methylester CO mode vibrations at 1696 and 1750 cm−1, respectively. In FRL-PSI the A–1 cofactor is a Chl f molecule with 131-keto and 133-methylester CO mode vibrations at 1702 and 1754 cm−1, respectively.

SF4 as the Fluorination Reactant for Al2O3 and VO2 Thermal Atomic Layer Etching

Thermal atomic layer etching (ALE) is an important technique for the precise isotropic etching of nanostructures. Thermal ALE of many materials can be achieved using a two-step fluorination and ligand-exchange reaction mechanism. Most previous thermal ALE processes have used HF as the fluorination reactant. Alternative fluorination reactants may be needed because HF is a weak nucleophilic fluorination reactant. Stronger fluorination agents may be required for the fluorination of some materials. To demonstrate the usefulness of SF4 as an alternative to HF, thermal Al2O3 ALE was compared using SF4 or HF together with Sn(acac)2 as the metal precursor for ligand exchange. SF4 and HF were observed to behave similarly as fluorination reactants during Al2O3 ALE. The mass gains during the initial SF4 and HF exposures on Al2O3 atomic layer deposition (ALD) films at 200 °C were comparable at 35 and 38 ng/cm2, respectively, using quartz crystal microbalance measurements. In addition, the etch rates were similar at 0.20 and 0.28 Å/cycle for Al2O3 ALE using SF4 and HF, respectively, at 200 °C. Thermal VO2 ALE was also performed for the first time using SF4 or HF and Sn(acac)2 as the reactants. There was evidence that SF4 is a stronger fluorination reactant than HF for VO2 fluorination. The mass gains during the initial SF4 and HF exposures on VO2 ALD films were 38 and 20 ng/cm2, respectively, at 200 °C. Thermal VO2 ALE also had a higher etch rate when fluorinating with SF4 compared with HF. Etch rates of 0.30 and 0.11 Å/cycle were measured for VO2 ALE using SF4 and HF, respectively, together with Sn(acac)2 at 200 °C. Fourier transform infrared experiments were also used to monitor fluorination of the Al2O3 and VO2 ALD films by SF4 or HF. FTIR difference spectroscopy was used to observe the increase of Al–F and V–F stretching vibrations and the loss of the Al–O and V—O/V═O stretching vibrations for Al2O3 and VO2, respectively, versus SF4 or HF exposure at 200 °C. Additional absorbance features after fluorination of the Al2O3 ALD films by SF4 were consistent with SFx surface species. SF4 is a useful fluorination agent for thermal ALE processes and can be used as an alternative to HF. In addition, SF4 may be necessary when fluorination requires a stronger fluorination reactant than HF.

Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.

The Opposite Anisotropic Piezoresistive Effect of ReS2.

Mechanical strain induced changes in the electronic properties of two-dimensional (2D) materials is of great interest for both fundamental studies and practical applications. The anisotropic 2D materials may further exhibit different electronic changes when the strain is applied along different crystalline axes. The resulting anisotropic piezoresistive phenomenon not only reveals distinct lattice-electron interaction along different principle axes in low-dimensional materials but also can accurately sense/recognize multidimensional strain signals for the development of strain sensors, electronic skin, human-machine interfaces, etc. In this work, we systematically studied the piezoresistive effect of an anisotropic 2D material of rhenium disulfide (ReS2), which has large anisotropic ratio. The measurement of ReS2 piezoresistance was experimentally performed on the devices fabricated on a flexible substrate with electrical channels made along the two principle axes, which were identified noninvasively by the reflectance difference microscopy developed in our lab. The result indicated that ReS2 had completely opposite (positive and negative) piezoresistance along two principle axes, which differed from any previously reported anisotropic piezoresistive effect in other 2D materials. We attributed the opposite anisotropic piezoresistive effect of ReS2 to the strain-induced broadening and narrowing of the bandgap along two principle axes, respectively, which was demonstrated by both reflectance difference spectroscopy and theoretical calculations.

The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.