0000000000555374
AUTHOR
Víctor A. Lórenz-fonfría
showing 13 related works from this author
A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes
2021
AbstractAccording 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 eq…
Vibrational and Molecular Properties of Mg2+ Binding and Ion Selectivity in the Magnesium Channel MgtE
2018
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-dire…
Photoluminescence study of terbium-exchanged ultrastable Y zeolites: Number of species, photoluminescence decays and decay-associated spectra
2008
Terbium-exchanged ultrastable Y (USY) zeolites were investigated by using time-resolved photoluminescence spectroscopy techniques and methods. To determine the distribution of terbium species in USY zeolites together with their photoluminescence properties, several analysis methods for the time-resolved luminescence spectra were used such as the area normalization of time-resolved photoluminescence spectra, singular value decomposition, global nonlinear least squares, and the maximum entropy. Except for a questionable long lifetime, small contribution of a terbium species with lifetime of 1.9¿2.1 ms, all the experimental and analysis results converged to a two terbium species distribution w…
Infrared Difference Spectroscopy of Proteins: From Bands to Bonds
2020
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) functional…
Orientation of non-spherical protonated water clusters revealed by infrared absorption dichroism
2018
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 amplifica…
Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1
2014
Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2(380) intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in…
pH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin
2017
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…
Potential Second-Harmonic Ghost Bands in Fourier Transform Infrared (FT-IR) Difference Spectroscopy of Proteins
2018
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…
Probing a Polar Cluster in the Retinal Binding Pocket of Bacteriorhodopsin by a Chemical Design Approach
2012
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 apoprotein…
Photoexcitation of the P4480 State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2
2018
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...
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
2014
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 num…
Retinal vibrations in bacteriorhodopsin are mechanically harmonic but electronically anharmonic: evidence from overtone and combination bands
2021
AbstractVibrations 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 stretch…
Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s
2017
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-res…