8 research outputs found
Photoexcitation of the P4480 state induces a secondary photocycle that potentially desensitizes channelrhodopsin-2
Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P3520 intermediate of the primary all-trans photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P4480, the last intermediate of the all-trans photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I3530. Our results indicate that the D2 and O2 states correspond to the P4480 and I3530 intermediates, connecting desensitization of ChR2 with the photochemical properties of the P4480 intermediate
Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s
We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800–1700 cm− 1) and on the CO rebinding reaction of myoglobin (1960–1840 cm− 1), at a spectral resolution of 1 cm− 1. The spectrometric setup covers the time range from 4 ns to nearly a second with a response time of 10–15 ns. Absorption changes as low as 1 × 10− 4 are detected in single-shot experiments at t > 1 μs, and of 5 × 10− 6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H2O)
Protein conformational changes and protonation dynamics probed by a single shot using quantum-cascade-laser-based IR spectroscopy
Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations
Orientation of non-spherical protonated water clusters revealed by infrared absorption dichroism
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
A photoswitchable helical peptide with light-controllable interface/transmembrane topology in lipidic membranes
The spontaneous insertion 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. A bottleneck for understanding these three steps is the lack of experimental approaches to perturb membrane-bound hydrophobic polypeptides out of equilibrium rapidly and reversibly. 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 reveals that trans KCALP-azo folds as a TM α-helix (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), the helical structure of KCALP-azo is maintained, but its helix tilt increased from 32 ± 5° to 79 ± 8°, indication of a reversible TM-to-MI transition. Further analysis indicates that this transition is incomplete, with cis KCALP-azo existing in a ∼90% TM and ∼10% MI mixture
Photoexcitation of the P<sub>4</sub><sup>480</sup> State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin‑2
Channelrhodopsins
(ChRs) are light-gated cation channels. In spite
of their wide use to activate neurons with light, the photocurrents
of ChRs rapidly decay in intensity under both continuous illumination
and fast trains of light pulses, broadly referred to as desensitization.
This undesirable phenomenon has been explained by two interconnected
photocycles, each of them containing a nonconductive dark state (D1
and D2) and a conductive state (O1 and O2). While the D1 and O1 states
correspond to the dark-state and P<sub>3</sub><sup>520</sup> intermediate
of the primary all-<i>trans</i> photocycle of ChR2, the
molecular identity of D2 and O2 remains unclear. We show that P<sub>4</sub><sup>480</sup>, the last intermediate of the all-<i>trans</i> photocycle, is photoactive. Its photocycle, characterized by time-resolved
UV/vis spectroscopy, contains a red-shifted intermediate, I<sub>3</sub><sup>530</sup>. Our results indicate that the D2 and O2 states correspond
to the P<sub>4</sub><sup>480</sup> and I<sub>3</sub><sup>530</sup> intermediates, connecting desensitization of ChR2 with the photochemical
properties of the P<sub>4</sub><sup>480</sup> intermediate
Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies
Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm−1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm−1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm−1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins
Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold
CatedresAltres ajuts: Ministerio de Universidades Margarita Salas Award (MGSD2021-10 to M.L.-M.); Universitat Autònoma De Barcelona Predoctoral Fellowship (B21P0033 to E.C.-H.); the Royal Society of Chemistry for Financial Support through a RS International Exchanges award (IES\R\193089 to C.D.); Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the collaborative research center 1507 “Membrane-associated Protein Assemblies, Machineries, and Supercomplexes” – Project ID 450648163 (to UAH); Cluster of Excellence “Balance of the Microverse” EXC2051 – Project-ID 390713860 (to UAH).TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target