Víctor A. Lórenz-Fonfría

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating.

Significance It was always a dream to control cells and living animals by light. Discovery of channelrhodopsin turned the dream into reality because this light-activated cation channel is able to elicit action potentials with unprecedented spatial and temporal resolution. To unravel the underlying molecular mechanism, we have applied time-resolved IR spectroscopy, and we suggest how the observed proton transfer and the protein conformational changes lead to opening of the cation channel. Our results will not only contribute to the rational design of channelrhodopsin variants with improved properties, but also help to decipher the temporal sequence in the gating of ion channels. 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 (P2390 and P3520) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t1/2 = 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 P4480 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.

Channelrhodopsin unchained: 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. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Lysyl Oxidase-like 2 Deaminates Lysine 4 in Histone H3

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.

Spectroscopic and Kinetic Evidence on How Bacteriorhodopsin Accomplishes Vectorial Proton Transport under Functional Conditions

Vectorial transport by pumps requires a switch in the accessibility (or affinity) of the ion binding site from the extracelullar to the cytoplamic side, or vice versa. In the proton-pump bacteriorhodopsin (bR) the nature of this switch mechanism is still controversial, although it is expected to occur during the transition between two M substates. Here, we characterized this transition by time-resolved Fourier transform infrared (FT-IR) spectroscopy under functional conditions, using a novel approach for the analysis of kinetic data: the regularized inversion of an eigenvalue problem. The use of IR spectroscopy allowed the simultaneous evaluation of the involvement of the protein backbone, retinal, amino acid side chains, and internal water molecules in the switch mechanism. We provide solid evidence that the switch is not associated with protein backbone conformational changes. On the other hand, changes in the retinal conformation (or in the orientation of the Schiff Base (SB) during the switch) are reasonably although not completely discarded. We found that the proton release group (PRG), a delocalized proton characterized by a broad continuum band in the infrared, deprotonates in the transition between two M substates. Vectorial proton transport is most likely guaranteed by the coupled proton affinity changes resulting from the PRG deprotonation, favoring an affinity-based over an accessibility-based switch mechanism.

Active Internal Waters in the Bacteriorhodopsin Photocycle. A Comparative Study of the L and M Intermediates at Room and Cryogenic Temperatures by Infrared Spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.

Maximum Entropy Deconvolution of Infrared Spectra: Use of a Novel Entropy Expression Without Sign Restriction

Absorbance and difference infrared spectra are often acquired aiming to characterize protein structure and structural changes of proteins upon ligand binding, as well as for many other chemical and biochemical studies. Their analysis requires as a first step the identification of the component bands (number, position, and area) and as a second step their assignment. The first step of the analysis is challenged by the habitually strong band overlap in infrared spectra. Therefore, it is useful to make use of a mathematical method able to narrow the component bands to the extent to eliminate, or at least reduce, the band overlap. Additionally, to be of general applicability this method should permit negative values for the solution. We present a maximum entropy deconvolution approach for the band-narrowing of absorbance and difference spectra showing the required characteristics, which uses the generalized negative Burg-entropy (Itakura–Saïto discrepancy) generalized for difference spectra. We present results on synthetic noisy absorbance and difference spectra, as well as on experimental infrared spectra from the membrane protein bacteriorhodopsin.

Structural insights into the activation mechanism of melibiose permease by sodium binding

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.

Bayesian Maximum Entropy (Two-Dimensional) Lifetime Distribution Reconstruction from Time-Resolved Spectroscopic Data

Time-resolved spectroscopy is often used to monitor the relaxation processes (or reactions) of physical, chemical, and biochemical systems after some fast physical or chemical perturbation. Time-resolved spectra contain information about the relaxation kinetics, in the form of macroscopic time constants of decay and their decay associated spectra. In the present paper we show how the Bayesian maximum entropy inversion of the Laplace transform (MaxEnt-iLT) can provide a lifetime distribution without sign-restrictions (or two-dimensional (2D)-lifetime distribution), representing the most probable inference given the data. From the reconstructed (2D) lifetime distribution it is possible to obtain the number of exponentials decays, macroscopic rate constants, and exponential amplitudes (or their decay associated spectra) present in the data. More importantly, the obtained (2D) lifetime distribution is obtained free from pre-conditioned ideas about the number of exponential decays present in the data. In contrast to the standard regularized maximum entropy method, the Bayesian MaxEnt approach automatically estimates the regularization parameter, providing an unsupervised and more objective analysis. We also show that the regularization parameter can be automatically determined by the L-curve and generalized cross-validation methods, providing (2D) lifetime reconstructions relatively close to the Bayesian best inference. Finally, we propose the use of MaxEnt-iLT for a more objective discrimination between data-supported and data-unsupported quantitative kinetic models, which takes both the data and the analysis limitations into account. All these aspects are illustrated with realistic time-resolved Fourier transform infrared (FT-IR) synthetic spectra of the bacteriorhodopsin photocycle.

Protein Fluctuations as the Possible Origin of the Thermal Activation of Rod Photoreceptors in the Dark

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.

The Role and Selection of the Filter Function in Fourier Self-Deconvolution Revisited

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.