Covalent Drug Binding in Live Cells Monitored by Mid-IR Quantum Cascade Laser Spectroscopy: Photoactive Yellow Protein as a Model System.

The detection of drug-target interactions in live cells enables analysis of therapeutic compounds in a native cellular environment. Recent advances in spectroscopy and molecular biology have facilitated the development of genetically encoded vibrational probes like nitriles that can sensitively report on molecular interactions. Nitriles are powerful tools for measuring electrostatic environments within condensed media like proteins, but such measurements in live cells have been hindered by low signal-to-noise ratios.

Structural Transition from Closed to Open for the Influenza A M2 Proton Channel as Observed by Proton-Detected Solid-State NMR.

The influenza A M2 protein is an acid-activated proton channel and an established pharmaceutical target for antiflu drugs. Here, we studied the conductance domain of the tetrameric M2 channel (construct 18-60) using proton-detected solid-state NMR under native-like conditions in lipid bilayers. We obtained results at different pH values relevant to the virus life cycle: pH 7.

Hydrogen-Producing Catalysts Based on Ferredoxin Scaffolds.

Current attempts to transform our fossil fuel-based society into a sustainable one involve learning from and employing the biochemistry of nature. The process of photosynthesis is exemplary for utilizing sunlight as a regenerative energy source. Enzymes like hydrogenases, which reduce protons to molecular hydrogen (H) under ambient conditions, are model biocatalysts for generating sustainable, clean fuels.

Label-Free Detection of Virus-Membrane Interactions Using Surface-Enhanced Infrared Absorption (SEIRA) Spectroscopy.

Viral binding and membrane fusion are essential steps in viral infection, mediated by viral proteins that bind to host cell receptors and facilitate the fusion between viral and host membranes. Targeting these steps for the development of new antiviral strategies requires methods that enable investigating virus-membrane interactions under in-situ conditions, while providing mechanistic insights on a molecular level. Here, we demonstrate the use of surface-enhanced infrared absorption (SEIRA) spectroscopy combined with tethered bilayer lipid membranes (tBLMs) for the label-free detection of virus-membrane interactions, using the Influenza A/X-31 virus (IAV) as a model.

Data-driven signal-to-noise enhancement in scattering near-field infrared microscopy.

This study introduces a machine-learning approach to enhance signal-to-noise ratios in scattering-type scanning near-field optical microscopy (s-SNOM). While s-SNOM offers a high spatial resolution, its effectiveness is often hindered by low signal levels, particularly in weakly absorbing samples. To address these challenges, we utilize a data-driven "patch-based" machine learning reconstruction method, incorporating modern generative adversarial neural networks (CycleGANs) for denoising s-SNOM images.

Hydrogen Bond Blueshifts in Nitrile Vibrational Spectra Are Dictated by Hydrogen Bond Geometry and Dynamics.

Vibrational Stark effect (VSE) spectroscopy has become one of the most important experimental approaches to determine the strength of noncovalent, electrostatic interactions in chemistry and biology and to quantify their influence on structure and reactivity. Nitriles (C≡N) have been widely used as VSE probes, but their application has been complicated by an anomalous hydrogen bond (HB) blueshift which is not encompassed within the VSE framework. We present an empirical model describing the anomalous HB blueshift in terms of H-bonding geometry, i.

Critical Evaluation of Polarizable and Nonpolarizable Force Fields for Proteins Using Experimentally Derived Nitrile Electric Fields.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins ( , (17), 7562-7567).

In Situ Spectroscopic Detection of Large-Scale Reorientations of Transmembrane Helices During Influenza A M2 Channel Opening.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function.

Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of β-Lactamases.

The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 β-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation.

Potential Distribution across Model Membranes.

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes.

A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active site.

The catalytic power of an electric field depends on its magnitude and orientation with respect to the reactive chemical species. Understanding and designing new catalysts for electrostatic catalysis thus requires methods to measure the electric field orientation and magnitude at the molecular scale. We demonstrate that electric field orientations can be extracted using a two-directional vibrational probe by exploiting the vibrational Stark effect of both the C=O and C-D stretches of a deuterated aldehyde.

Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.

Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to H-bonding and non-H-bonding interactions.

The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete .

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (Rh1) from the smut pathogen the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle.

The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases.

The interplay of enzyme active site electrostatics and chemical positioning is important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 β-lactamase to TEM-52 toward extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and noncovalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2.

Quantification of Local Electric Field Changes at the Active Site of Cytochrome Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations.

Cytochrome oxidase (CO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CO from .

Testing the Limitations of MD-Based Local Electric Fields Using the Vibrational Stark Effect in Solution: Penicillin G as a Test Case.

Noncovalent interactions underlie nearly all molecular processes in the condensed phase from solvation to catalysis. Their quantification within a physically consistent framework remains challenging. Experimental vibrational Stark effect (VSE)-based solvatochromism can be combined with molecular dynamics (MD) simulations to quantify the electrostatic forces in solute-solvent interactions for small rigid molecules and, by extension, when these solutes bind in enzyme active sites.

Biosynthetic Incorporation of Site-Specific Isotopes in β-Lactam Antibiotics Enables Biophysical Studies.

A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Toward this goal, we developed a biosynthetic strategy for the site-specific incorporation of C labels into the canonical β-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C.

Vibrational Stark Spectroscopy of Fluorobenzene Using Quantum Cascade Laser Dual Frequency Combs.

We demonstrate the performance of a dual frequency comb quantum cascade laser (QCL) spectrometer for the application of vibrational Stark spectroscopy. Measurements performed on fluorobenzene with the dual-comb spectrometer (DCS) were compared to results obtained using a conventional Fourier transform infrared (FT-IR) instrument in terms of spectral response, parameter estimation, and signal-to-noise ratio (S/N). The dual-comb spectrometer provided similar qualitative and quantitative data as the FT-IR setup in 250 times shorter acquisition time.

The C-Terminal VPRTES Tail of LL-37 Influences the Mode of Attachment to a Lipid Bilayer and Antimicrobial Activity.

Cathelicidins are a family of host defense antimicrobial peptides in mammalian species. Among them, LL-37 is the only peptide of this family found in humans. Although LL-37 has been intensively investigated in the past, the mode of exerting its bactericidal activity through the specific interactions with bacterial membranes remains elusive.

On the pH-Modulated Ru-Based Prodrug Activation Mechanism.

The Ru-based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, Ru and Ru, was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent.

Long-Range Modulations of Electric Fields in Proteins.

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification.

In Situ Spectroelectrochemical Studies into the Formation and Stability of Robust Diazonium-Derived Interfaces on Gold Electrodes for the Immobilization of an Oxygen-Tolerant Hydrogenase.

Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation.

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations.