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Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.

The spontaneous (translocon-unassisted) folding/insertion of helical transmembrane (TM) protein fragments into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion [1]. Understanding the molecular mechanism underlaying these steps has been challenged by the lack of suitable experimental approaches to perturb protein fragments rapidly and reversibly out of equilibrium once reconstituted in membranes. We have been actively working in implementing a strategy to control/perturb the folding and membrane topology of protein fragments with light. We have started with a 24-residues-long hydrophobic -helical polypeptide from the KALP family as a model for a helical transmembrane protein fragment. We introduced two Cys to KALP and coupled it to an azobenzene photoswitch (KCALP-azo), as done before for helical soluble protein fragments [2]. By polarized FTIR spectroscopy we determined that KCALP-azo (trans azobenzene) folds as a TM -helix, as KALP does, with a helix tilt angle of 32 ± 5. After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), we confirmed the optical control of the membrane topology of KCALP-azo: its helix tilt changes reversibly from 32 ± 5° (TM topology, blue light) to 79 ± 8° (MI topology, UV light) [3].We have started to use short UV and blue light pulses in combination with time-resolved FTIR difference spectroscopy [4]. These experiments exploit the here achieved optical control to provide a detailed picture on how a membrane interface helical fragment inserts in lipidic membrane, a relevant information currently only available from MD simulations [5].

Vibrations of the chromophore in the membrane protein bacteriorhodopsin (BR), a protonated Schiff base retinal, have been studied for decades, both by resonance Raman and by infrared (IR) difference spectroscopy. In spite the light-induced IR difference spectrum between the K intermediate (13-cis retinal) and the initial BR state (all-trans retinal) being first published almost 40 years ago, we present here unreported bands in the 2500 to 1800 cm−1 region. We show that the bands between 2500 and 2300 cm−1 originate from overtone and combination transitions of retinal C-C stretches. We assigned some of the newly reported bands below 2300 cm−1 to the combination of retinal C-C stretches with methyl rocks and with hydrogen-out-of-plane vibrations. Remarkably, experimental C-C overtone bands appeared at roughly twice the wavenumber of their fundamentals, with anharmonic mechanical constants ≤ 3.5 cm−1, and in some cases of ∼1 cm−1. Comparison of combination and fundamental bands indicates that most of the mechanical coupling constants are also very small. Despite the mechanical quasi-harmonicity of the C-C stretches, the area of their overtone bands was only ∼50 to ∼100 times smaller than of their fundamental bands. We concluded that electronic anharmonicity, the second mechanism giving intensity to overtone bands, must be particularly high for the retinal C-C stretches. We corroborated the assignments of negative bands in the K-BR difference spectrum by ab initio anharmonic spectral calculations of all-trans retinal in BR, which also reproduced reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electronic anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4 to 20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations.

According to the three-step model, the spontaneous insertion and folding of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. However, understanding these three steps with molecular detail has been challenged by the lack of suitable experimental approaches to rapidly and reversibly perturb membrane-bound hydrophobic polypeptides out of equilibrium. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy shows that dark-adapted KCALP-azo (trans azobenzene) folds as a TM α-helix, with its central TM region displaying an average tilt of 36 ± 4° with the membrane normal (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), spectral changes by FTIR spectroscopy indicate that the helical structure of KCALP-azo is maintained but the peptide experiences a more polar environment. Interestingly, pH changes induced similar spectral alterations in the helical peptide LAH4, with a well-characterized pH-dependent TM/MI equilibrium. Polarized experiments confirmed that the membrane topology of KCALP-azo is altered by light, with its helix tilt changing reversibly from 32 ± 5° (TM topology, blue light) to 79 ± 8° (MI topology, UV light). Further analysis indicates that, while the trans isomer of KCALP-azo is ~100% TM, the cis isomer exists in a ~90% TM and ~10% MI mixture. Strategies to further increase the perturbation of the TM/MI equilibrium with the light are briefly discussed.

Magnesium ions (Mg2+) are crucial for various biological processes. A bacterial Mg2+ channel, MgtE, tightly regulates the intracellular Mg2+ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg2+ sensor. The ion selectivity for Mg2+ over Ca2+ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg2+ and Ca2+. Using site-directed mutagenesis, vibrational bands at 1421 (Mg2+), 1407 (Mg2+), ∼1440 (Ca2+), and 1390 (Ca2+) cm–1 were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of th...

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 illum...

Fourier transform infrared (FT-IR) difference absorption spectroscopy is a common method for studying the structural and dynamical aspects behind protein function. In particular, the 2800–1800 cm−1 spectral range has been used to obtain information about internal (deuterated) water molecules, as well as site-specific details about cysteine residues and chemically modified and artificial amino acids. Here, we report on the presence of ghost bands in cryogenic light-induced FT-IR difference spectra of the protein bacteriorhodopsin. The presence of these ghost bands can be particularly problematic in the 2800–1900 cm−1 region, showing intensities similar to O–D vibrations from water molecules. We demonstrate that they arise from second harmonics from genuine chromophore bands located in the 1400–850 cm−1 region, generated by double-modulation artifacts caused from reflections of the IR beam at the sample and at the cryostat windows back to the interferometer (inter-reflections). The second-harmonic ghost bands can be physically removed by placing an optical filter of suitable cutoff in the beam path, but at the cost of losing part of the multiplexing advantage of FT-IR spectroscopy. We explored alternatives to the use of optical filters. Tilting the cryostat windows was effective in reducing the intensity of the second harmonic artifacts but tilting the sample windows was not, presumably by their close proximity to the focal point of the IR beam. We also introduce a simple numerical post-processing approach that can partially, but not fully, correct for second-harmonic ghost bands in FT-IR difference spectra.

Infrared continuum bands that extend over a broad frequency range are a key spectral signature of protonated water clusters. They are observed for many membrane proteins that contain internal water molecules, but their microscopic mechanism has remained unclear. Here we compute infrared spectra for protonated and unprotonated water chains, discs, and droplets from ab initio molecular dynamics simulations. The continuum bands of the protonated clusters exhibit significant anisotropy for chains and discs, with increased absorption along the direction of maximal cluster extension. We show that the continuum band arises from the nuclei motion near the excess charge, with a long-ranged amplification due to the electronic polarizability. Our experimental, polarization-resolved light–dark difference spectrum of the light-driven proton pump bacteriorhodopsin exhibits a pronounced dichroic continuum band. Our results suggest that the protonated water cluster responsible for the continuum band of bacteriorhodopsin is oriented perpendicularly to the membrane normal., Protein-bound water clusters play a key role for proton transport and storage in molecular biology. Here, the authors show by simulations and experiments that the orientation of non-spherical protonated water clusters in bacteriorhodopsin is unveiled by polarization-resolved infrared spectroscopy.

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm−1 to 1738 cm−1 in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm−1 in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm−1 and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na+-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na+ and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200–330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na+ ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na+ ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na+ binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na+ binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.

Light-gated ion permeation by channelrhodopsin-2 (ChR2) relies on the photoisomerization of the retinal chromophore and the subsequent photocycle, leading to the formation (on-gating) and decay (off-gating) of the conductive state. Here, we have analyzed the photocycle of a fast-cycling ChR2 variant (E123T mutation, also known as ChETA), by time-resolved UV/vis, step-scan FT-IR, and tunable quantum cascade laser IR spectroscopies with nanosecond resolution. Pre-gating conformational changes rise with a half-life of 200 ns, silent to UV/vis but detected by IR spectroscopy. They involve changes in the peptide backbone and in the H-bond of the side chain of the critical residue D156. Thus, the P1(500) intermediate must be separated into early and late states. Light-adapted ChR2 contains a mixture of all-trans and 13-cis retinal in a 70:30 ratio which are both photoactive. Analysis of ethylenic and fingerprint vibrations of retinal provides evidence that the 13-cis photocycle recovers in 1 ms. This recovery is faster than channel off-gating and most of the proton transfer reactions, implying that the 13-cis photocycle is of minor functional relevance for ChR2.

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm−1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin.

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).

Vibrational circular dichroism (VCD) spectra of aqueous solutions of proline were recorded in the course of titrations from basic to acidic pH using a spectrometer equipped with a quantum cascade laser (QCL) as an infrared light source in the spectral range from 1320 to 1220 cm(-1). The pH-dependent spectra were analyzed by singular value decomposition and global fitting of a two-pK Henderson-Hasselbalch model. The analysis delivered relative fractions of the three different protonation species. Their agreement with the relative fractions obtained from performing the same analysis on pH-dependent Fourier transform infrared (FT-IR) and QCL-IR spectra validates the quantitative results from QCL-VCD. Global fitting of the pH-dependent VCD spectra of L-proline allowed for extraction of pure spectra corresponding to anionic, zwitterionic, and cationic L-proline. From a static experiment, only pure spectra of the zwitterion would be accessible in a straightforward way. A comparison to VCD spectra calculated for all three species led to assignment of vibrational modes that are characteristic for the respective protonation states. The study demonstrates the applicability of QCL-VCD both for quantitative evaluation and for qualitative interpretation of dynamic processes in aqueous solutions.

Changes in vibrational circular dichroism (VCD) were recorded on-line during a chemical reaction. The chiral complex nickel-(–)-sparteine chloride was hydrolyzed to free (–)-sparteine base in a biphasic system of sodium hydroxide solution and chloroform (CHCl3). Infrared (IR) and VCD spectra were iteratively recorded after pumping a sample from the CHCl3 phase through a lab-built VCD spectrometer equipped with a tunable mid-IR quantum cascade laser light source, which allows for VCD measurements even in the presence of strongly absorbing backgrounds. Time-dependent VCD spectra were analyzed by singular value decomposition and global exponential fitting. Spectral features corresponding to the complex and free (–)-sparteine could be clearly identified in the fitted amplitude spectrum, which was associated with an exponential decay with an apparent time constant of 127 min (t½ = 88 min). Chirality 26:490–496, 2014. © 2014 Wiley Periodicals, Inc.

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H(+), Na(+) or Li(+) to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na(+) in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation. Database The GEO accession number for the data referred to this paper is GSE35600.

Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.

Methylation of lysine 4 (K4) within histone H3 has been linked to active transcription and is removed by LSD1 and the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here, we describe the deamination catalyzed by Lysyl oxidase-like 2 protein (LOXL2) as an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, LOXL2 activity is linked with the transcriptional control of CDH1 gene by regulating H3K4me3 deamination. These results reveal another H3 modification and provide a different mechanism for H3K4 modification.

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H + , Na + , or Li + . In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na + binding. Secondly, though Asp124 is not essential for Na + binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na + -induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na + binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.

Efficient retinal photoisomerization, signal transduction, and amplification contribute to single-photon electrical responses in vertebrates visual cells. However, spontaneous discrete electrical signals arising in the dark, with identical intensity and time profiles as those generated by genuine single photons (dark events), limit the potential capability of the rod visual system to discern single photons from thermal noise. It is accepted that the light and the thermal activation of the rod photoreceptor rhodopsin (Rho) triggers the light and the dark events, respectively. However the activation barrier for the dark events (80-110 kJ/mol) appears to be only half of the barrier for light-dependent activation of Rho (> or =180 kJ/mol). On the basis of these observations, it has been postulated that both processes should follow different pathways, but the molecular mechanism for the thermal activation process still remains an open question and subject of debate. Here, performing infrared difference spectroscopy measurements, we found that the -OH group of Thr118 from bovine Rho exhibits a slow but measurable hydrogen/deuterium exchange (HDX) under native conditions. Given the location of Thr118 in the X-ray structures, isolated from the aqueous phase and in steric contact with the buried retinal chromophore, we assume that a protein structural fluctuation must drive the retinal binding pocket (RBP) transiently open. We characterized the kinetics (rate and activation enthalpy) and thermodynamics (equilibrium constant and enthalpy) of this fluctuation from the global analysis of the HDX of Thr118-OH as a function of the temperature and pH. In parallel, using HPLC chromatography, we determined the kinetics of the thermal isomerization of the protonated 11-cis retinal in solution, as a model for retinal thermal isomerization in an open RBP. Finally, we propose a quantitative two-step model in which the dark activation of Rho is triggered by thermal isomerization of the retinal in a transiently opened RBP, which accurately reproduced both the experimental activation barrier and the rate of the dark events. We conclude that the absolute sensitivity threshold of our visual system is limited by structural fluctuations of the chromophore binding pocket rather than in the chromophore itself.

We introduce a method for the estimation of the mean Lorentzian bandwidth of the component bands in a spectrum. The method is computationally simple, using only the module of the Fourier transform of the spectrum, and its first derivative. Moreover, the presented method does not require knowledge of the number of bands in the spectrum, their band positions, or their band areas. Furthermore, it works on spectra containing Lorentzian bands, as well as Gaussian and Voigtian bands. Therefore, the introduced method seems especially well suited for obtaining a representative Lorentzian width for highly overlapped bands, independent of their number and Lorentzian/Gaussian character. We describe how different experimental limitations (spectral truncation, offset error, presence of noise, etc.) may affect the performance of the method, and when required we propose effective alternatives to minimize their effects. Finally, we show the application of the method to an experimental spectrum: the amide I band of a dry film of the solubilized ADP/ATP carrier. The estimation of the mean Lorentzian width can allow, for instance, for a more objective selection of the deconvolution width in Fourier self-deconvolution, allowing for a more objective and reliable analysis of the amide I band of proteins. The mean Lorentzian width can also be useful to obtain an estimation of the homogenous broadening and vibrational relaxation of the amide I vibration of proteins, without requiring complex pump-probe experiments.

The iron(III) azido complex of 5,10,15,20-tetraisopropylporphyrin was characterized with NMR, EPR, Mossbauer, and magnetic susceptibility. These physical methods indicate mixing of the high (S = 5/2) and intermediate (S = 3/2) spin-states of the iron atom. The results were interpreted in terms of the core contraction after nonplanar deformation of porphyrin ring by the bulky isopropyl substituents. In the IR spectrum of this complex, there are two signals at 2062 and 2048 cm -1 due to the antisymmetric vibration of the coordinated azido ligand. The split IR bands demonstrate that the two spin isomers are present, and that the S = 5/2 and 3/2 transition occurs sufficiently slow on the IR timescale. This is in remarkable contrast with the homogeneous spin-mixing model proposed for the S = 5/2 and 3/2 system. The present observations further suggests that the three S = 5/2, 3/2, and 1/2 states in iron(III) porphyrin commonly mix through thermal spin equilibrium.

Terbium-exchanged BEA zeolites were hydrophobized with phenyl-, vinyl-, and hexadecyltrimethoxysilanes by means of postsynthesis grafting. These materials were investigated using XRD, FT-IR, TGA, physical adsorption, and photoluminescence. Different methods for the analysis of the non-exponential decay of terbium photoluminescence in BEA zeolites were used ranging from discrete exponential to more complex approaches based on maximum entropy and global analysis. Two groups of decay times varying between 480 and 580 micros and 1-1.3 ms were assigned to the lifetimes of terbium exposed to water (unprotected) and protected by the organic groups, respectively. Our results showed that the preservation of terbium PL properties against detrimental effects of moisture adsorption could be ordered in the following sequence: hexadecylphenyl approximately vinyl. The photoluminescence results were in good agreement with the FT-IR, TGA, and physical adsorption data.

The number, position, area, and width of the bands in a lifetime distribution give the number of exponentials present in time-resolved data and their time constants, amplitudes, and heterogeneities. The maximum entropy inversion of the Laplace transform (MaxEnt-iLT) provides a lifetime distribution from time-resolved data, which is very helpful in the analysis of the relaxation of complex systems. In some applications both positive and negative values for the lifetime distribution amplitudes are physical, but most studies to date have focused on positive-constrained solutions. In this work, we first discuss optimal conditions to obtain a sign-unrestricted maximum entropy lifetime distribution, i.e., the selection of the entropy function and the regularization value. For the selection of the regularization value we compared four methods: the χ2 criterion and Bayesian inference (already used in sign-restricted MaxEnt-iLT), and the L-curve and the generalized cross-validation methods (not yet used in MaxEnt-iLT to our knowledge). Except for the frequently used χ2 criterion, these methods recommended similar regularization values, providing close to optimum solutions. However, even when an optimal entropy function and regularization value are used, a MaxEnt lifetime distribution will contain noise-induced errors, as well as systematic distortions induced by the entropy maximization (regularization-induced errors). We introduce the concept of the apparent resolution function in MaxEnt, which allows both the noise and regularization-induced errors to be estimated. We show the capability of this newly introduced concept in both synthetic and experimental time-resolved Fourier transform infrared (FT-IR) data from the bacteriorhodopsin photocycle.

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

The visual pigment rhodopsin has been extensively studied for the kinetics of its photointermediates by various spectroscopic methods. Unlike such archaeal retinal proteins as bacteriorhodopsin, visual rhodopsin does not thermally recover its dark state after photoexcitation, which precludes repeated excitation of a single sample and thereby complicates time-resolved experiments. Kinetic data on the late rhodopsin photointermediates have so far been available mainly from time-resolved ultraviolet (UV)-visible spectroscopy, but not from Fourier transform infrared (FTIR) spectroscopy. The latter has the advantage of being informative of structural changes of both chromophore and protein, but does not allow the highly reproducible, automated sample exchange procedures available to UV-visible spectroscopy. Using rapid-scan FTIR difference spectroscopy, we obtained time-resolved data sets that were analyzed by a maximum entropy inverse Laplace-transform. Covering the time range from 8 ms to 15 s at temperatures of 0 and −7°C, the transitions from the Lumi to the Meta I and from the Meta I to the Meta II photoproduct states could be resolved. In the transition from Meta I to Meta II, our data reveal a partial deprotonation of the retinal Schiff base preceding the conformational change of the receptor protein to Meta II. The technique and the results are discussed in regard to its advantages as well as its limitations. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 159–169, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.

We present a general method for curve-fitting Fourier manipulated spectra, comprising apodized, smoothed, derivatised and deconvoluted spectra. The analytical expressions of Fourier manipulated bands in the spectral domain, needed for the curve-fitting, are usually very complex or do not even exist; hence an accurate curve-fit of Fourier manipulated spectra becomes unfeasible. Our strategy is to construct both the model and their derivatives in the Fourier domain, where they have simple and general expressions, and then Fourier transform them back to the spectral domain. The first benefit of this approach is the accurate curve-fitting of Fourier deconvoluted spectra, a main step in the secondary structure estimation of proteins by FTIR spectroscopy.

The accessibility ofEscherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H+, Na+, melibiose). Analysis of the amide II band upon2H2O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H+·melibiose·MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on β-sheet accessibility, with the complex H+·melibiose·MelB being the most protected state against exchange, followed by Na+·melibiose·MelB. Although of smaller magnitude, similar changes in α-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly β-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4–5 and 10–11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.

The effect of Fourier deconvolution on band narrowing and on the decrease of signal-to-noise ratio has been studied for a generalized case in which the width used in the deconvolution does not match the actual bandwidth. For the identification of underlying component bands, our results show that application of infra-deconvolution (i.e., the bandwidth used for deconvolution is lower than the actual bandwidth) produces a high degradation of the signal-to-noise ratio and poor band narrowing. On the contrary, self- or over-deconvolution, with lower signal-to-noise degradation and higher band narrowing, seem more suitable for this purpose. Relative to quantitative analysis, we rely on both theoretical and practical aspects to propose the generalized use of Voigt band shapes as a fairly correct general model to be used in the curve fitting of deconvoluted bands. With its use, the curve fitting of noise-free deconvoluted bands retrieved the original band parameters with high accuracy. The noise effect on the parameter precision obtained by curve fitting a deconvoluted noisy Lorentzian band was also studied. Finally, the existence of optimum deconvolution parameters for curve fitting complex spectra is considered, and a general recommendation for approaching this optimum is given.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With -10(2)-10(3) repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as -10(-) (4), sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P2380) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.

Changes in vibrational circular dichroism (VCD) were recorded on-line during a chemical reaction. The chiral complex nickel-(-)-sparteine chloride was hydrolyzed to free (-)-sparteine base in a biphasic system of sodium hydroxide solution and chloroform (CHCl(3)). Infrared (IR) and VCD spectra were iteratively recorded after pumping a sample from the CHCl(3) phase through a lab-built VCD spectrometer equipped with a tunable mid-IR quantum cascade laser light source, which allows for VCD measurements even in the presence of strongly absorbing backgrounds. Time-dependent VCD spectra were analyzed by singular value decomposition and global exponential fitting. Spectral features corresponding to the complex and free (-)-sparteine could be clearly identified in the fitted amplitude spectrum, which was associated with an exponential decay with an apparent time constant of 127 min (t(½) = 88 min).

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 10−12s 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.

The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P-2(390) and P-3(520)) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane a-helices with a t(1/2) = 60 mu s, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t(1/2) = 10 mu s), with the latter being reprotonated by aspartic acid 156 (t(1/2) = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P-4(480) state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.

(Molecular Cell 46, 369–376; May 11, 2012)We, the authors, are retracting this article because of inappropriate manipulation of the data in the H3K4me3 blot in the left panel of Figure 3C (LOXL2). Specifically, a duplicated/inverted lane 3 replaced the original lane 1.Readers raised concerns about data presented in Figures 1, 3, and 4. We provided all of the raw data corresponding to these figures to Molecular Cell and the Scientific Integrity Committee of our institute, the IMIM (Hospital del Mar Medical Research Institute). The journal and the Scientific Integrity Committee of the IMIM found several issues, but determined that apart from the instance in Figure 3C, there was no evidence of intentional misconduct. However, because the data in the H3K4me3 blot in the left (LOXL2) panel of Figure 3C were inappropriately manipulated, resulting in a figure that does not accurately represent the data as obtained, we are retracting the paper.We wish to note that other experimental replicates reproduced the result in the H3K4me3 blot in Figure 3C’s left (LOXL2) panel. In addition, our subsequent papers showing that oxidation of H3 by LOXL2 has a central role in EMT (Millanes-Romero et al., Mol. Cell 2013) and that LOXL2 oxidizes methylated TAF10 (Iturbide et al., Mol. Cell 2015) support the central findings of this paper.We apologize to the scientific community for any inconveniences resulting from the publication and retraction of this manuscript.

The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IR(diff)) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IR(diff) spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na(+)-induced ATR-IR(diff) spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IR(diff)) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IR(diff) spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na(+)-induced ATR-IR(diff) spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.

We present a general approach for probing the kinetics of specific molecular processes in proteins by time-resolved Fourier transform infrared (IR) spectroscopy. Using bacteriorhodopsin (bR) as a model we demonstrate that by appropriately monitoring some selected IR bands it is possible obtaining the kinetics of the most important events occurring in the photocycle, namely changes in the chromophore and the protein backbone conformation, and changes in the protonation state of the key residues implicated in the proton transfers. Besides confirming widely accepted views of the bR photocycle, our analysis also sheds light into some disputed issues: the degree of retinal torsion in the L intermediate to respect the ground state; the possibility of a proton transfer from Asp85 to Asp212; the relationship between the protonation/deprotonation of Asp85 and the proton release complex; and the timing of the protein backbone dynamics. By providing a direct way to estimate the kinetics of photocycle intermediates the present approach opens new prospects for a robust quantitative kinetic analysis of the bR photocycle, which could also benefit the study of other proteins involved in photosynthesis, in phototaxis, or in respiratory chains.

Time-resolved luminescence spectra of europium in parent and silylated microporous-mesoporous materials were analyzed by using the maximum entropy method and global multi-exponential fitting, providing lifetime distributions and decay-associated spectra. Silylation was used as a method for the hydrophobization of the materials surface in order to inhibit the moisture intrusion at the lanthanide's sites. Due to the well-known sensitivity of the europium photoluminescence properties to the local environment, our approach can simplify the description of the complex structure-photoluminescence relationships in terms of the individual europium species that contribute to the total emission of the system.

Overlapped bands often appear in applications of infrared spectroscopy, for instance in the analysis of the amide I band of proteins. Fourier self-deconvolution (FSD) is a popular band-narrowing mathematical method, allowing for the resolution of overlapped bands. The filter function used in FSD plays a significant role in the factor by which the deconvolved bands are actually narrowed (the effective narrowing), as well as in the final signal-to-noise degradation induced by FSD. Moreover, the filter function determines, to a good extent, the band-shape of the deconvolved bands. For instance, the intensity of the harmful side-lobule oscillations that appear in over-deconvolution depends importantly on the filter function used. In the present paper we characterized the resulting band shape, effective narrowing, and signal-to-noise degradation in infra-, self-, and over-deconvolution conditions for several filter functions: Triangle, Bessel, Hanning, Gaussian, Sinc2, and Triangle2. We also introduced and characterized new filters based on the modification of the Blackmann filter. Our conclusion is that the Bessel filter (in infra-, self-, and mild over-deconvolution), the newly introduced BL3 filter (in self- and mild/moderate over-deconvolution), and the Gaussian filter (in moderate/strong over-deconvolution) are the most suitable filter functions to be used in FSD.

Terbium-exchanged ZSM-5, MOR and (H)BEA zeolites were silylated with phenyl-, vinyl- and hexadecyl trimethoxysilanes via a post-synthesis grafting. All samples were investigated by means of PXRD, FT-IR, TGA, physical adsorption, DR-UV-Vis and time-resolved photoluminescence spectroscopy. From the comparison of the photoluminescence decays of terbium-exchanged in parent (non-silylated) and silylated zeolites, it resulted that the silylation efficiency of the various alkoxysilanes is determined by the type of zeolite and follows the sequences: phenyl > vinyl > hexadecyl > parent for ZSM-5, hexadecyl a parts per thousand phenyl a parts per thousand vinyl > parent for MOR and hexadecyl > phenyl a parts per thousand vinyl > a parts per thousand parent for BEA zeolites, respectively.

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro91 and Pro186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8kcal/mol, respectively, compared to 115.8kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.