Biology subjects

Lorenz-Fonfria, V. A.

Publications and source records attributed to Lorenz-Fonfria, V. A..

4 recordsLinked to original sources

bioRxiv2026

Membrane Lipid Composition and Asymmetry Act as a Switch for Cell-Penetrating Peptide Translocation versus Leakage

Cell-penetrating peptides (CPPs) can enable intracellular access while avoiding cytotoxicity, yet their behavior is highly sensitive to membrane composition. Here we show that lipid composition and leaflet asymmetry act as a switch that determines whether the amphipathic CPP MAP undergoes non-disruptive translocation or stabilizes membrane pores that drive leakage. Using computational electrophysiology (CompEL) simulations in membranes of increasing physiological relevance, we find that symmetric anionic bilayers favor MAP insertion coupled to transmembrane pore stabilization in POPC:POPG membranes, reproducing progressive dye release in liposome leakage assays. Incorporation of cholesterol reduces the number of inserted peptides yet enhances pore stabilization, consistent with faster leakage kinetics in cholesterol-containing vesicles. In contrast, an asymmetric membrane model mimicking the eukaryotic plasma membrane (POPC outer leaflet; POPC:POPS inner leaflet) supports MAP translocation without sustained membrane disruption. This delivery-relevant mechanism is supported by efficient MAP internalization in HEK293 cells while maintaining high viability. Polarized ATR-FTIR provides structural context, indicating predominantly membrane-associated -helical conformations across lipid compositions. Together, these results establish lipid asymmetry and composition as actionable biointerface parameters that tune CPP function between translocation and leakage and demonstrate an experimentally benchmarked framework for predicting membrane outcomes across complex lipid environments.

biophysics↗

bioRxiv2022

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of -helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A extension of the LOV domain in controlling EL222 photocycle length. SignificanceThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C{equiv}N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.

biophysics↗

bioRxiv2021

Retinal vibrations in bacteriorhodopsin are mechanically harmonic but electronically anharmonic: evidence from overtone and combination bands

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.

biophysics↗

bioRxiv2021

A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

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 -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 -helix, with its central TM region displaying an average tilt of 36 {+/-} 4{degrees} 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{degrees} (TM topology, blue light) to 79 {+/-} 8{degrees} (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.

biophysics↗