Victor Armando Lorenz Fonfria

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 nondisruptive translocation or stabilizes membrane pores that drive leakage. Using high-field computational electrophysiology (CompEL) simulations in membranes of increasing physiological relevance, we show that electrically induced membrane defects allow the insertion of MAP peptides. We find 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 and conducts to faster leakage kinetics in cholesterol-containing vesicles. In contrast, a minimal asymmetric membrane model (POPC extracellular leaflet; POPC/POPS intracellular 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 α-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.

Absorption spectra of aqueous samples measured by transmission need to be acquired using very thin cells (5-50 μm) when targeting the mid-infrared (mid-IR) region, due to the strong background absorbance of liquid water. The thickness of the cell used controls the pathlength of the light through the sample, a value needed for instance to transform absorption spectra into molar absorption coefficient spectra, or to determine solute concentrations from absorption spectra. The most accurate way to determine the thickness of a thin empty cell (i.e., filled with air) is from the period of an interference pattern, known as interference fringes, that arises when the cell is placed perpendicular to the path of light in the spectrometer. However, this same approach is not directly applicable to determine the thickness of a cells filled with an aqueous solution, due partially to the smaller amplitude of the interference fringes but fundamentally caused by its complex waveform, with a wavenumber-dependent oscillation period. Here, using Fresnel equations, we derived analytical expressions to model interference fringes in absorption spectra obtained by transmission, valid also for aqueous samples. We also present a novel Fourier-based analysis of the interference fringes that, in combination with the derived analytical expressions, allowed us to determine the pathlength of aqueous samples with an error below 50 nm. We implemented this novel approach to analyze interference fringes as a Live Script running in the software Matlab. As an application, we measured absorption spectra of a 97 mM solution of MES buffer (at low and high pH) using cells of various nominal thicknesses (6, 25 and 50 μm). The molar absorption coefficient spectrum for the acidic and basic forms of MES, worked out using pathlengths values determined using the present approach, were virtually identical regardless of the nominal thickness of the cells used, indicating that the thickness determined for the cells were consistent and likely very accurate. These results illustrate the performance of the presented method to determine the pathlength of aqueous samples, as well as for its utility to obtain accurate molar absorption coefficient spectra of water-soluble molecules in the mid-IR region.

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 45 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'α helix interface (CNF31 and CNF35) precede FMN‐cysteinyl adduct scission, folding of α‐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'α extension of the LOV domain in controlling EL222 photocycle length.

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.

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’α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-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’α extension of the LOV domain in controlling EL222 photocycle length. Significance: The 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≡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.

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.

Fundamental 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. Such studies started comparing vibrational changes between the initial BR state (all-trans retinal) and the K intermediate (13-cis retinal), being later extended to the rest of intermediates. They contributed to our understanding of the proton-pumping mechanism of BR by exploiting the sensitivity of fundamental vibrational transitions of the retinal to its conformation. Here, we report on new bands in the 2,500 to 1,800 cm⁻¹ region of the K-BR difference FT-IR spectrum. We show that the bands between 2,500 and 2,300 cm⁻¹ originate from overtone and combination transitions from C-C stretches of the retinal. We assigned bands below 2,300 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 electrical 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 FT-IR spectrum by ab initio anharmonic vibrational calculations of all-trans retinal in BR using a quantum-mechanics/molecular mechanics approach, reproducing reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electrical anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4–20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations. The relatively intense overtone and combination bands of the retinal might open the possibility to detect retinal conformational changes too subtle to significantly affect fundamental transitions but leaving a footprint in overtone and combination transitions.

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.