Biology subjects

Kozuch, J.

Publications and source records attributed to Kozuch, J..

3 recordsLinked to original sources

bioRxiv2025

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. In this study, we design a spectrometer based on a double-beam quantum cascade laser (QCL)-based transmission infrared (IR) source with balanced detection that can significantly enhance sensitivity to nitrile vibrational probes embedded in proteins within cells compared to a conventional FTIR spectrometer. Using this approach, we detect small-molecule binding in E. coli, with particular focus on the interaction between para-coumaric acid (pCA) and nitrile-incorporated photoactive yellow protein (PYP). This system effectively serves as a model for investigating covalent drug binding in a cellular environment. Notably, we observe large spectral shifts of up to 15 cm-1 for nitriles embedded in PYP between the unbound and drug-bound states directly within bacteria, in agreement with observations for purified proteins. Such large spectral shifts are ascribed to the changes in the hydrogen-bonding environment around the local environment of nitriles, accurately modeled through high-level molecular dynamics simulations using the AMOEBA force field. Our findings underscore the QCL spectrometers ability to enhance sensitivity for monitoring drug-protein interactions, offering new opportunities for advanced methodologies in drug development and biochemical research. Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journals Instructions for Authors for TOC graphic specifications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/670201v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13bd30forg.highwire.dtl.DTLVardef@9db2bcorg.highwire.dtl.DTLVardef@141590forg.highwire.dtl.DTLVardef@13dfcf2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

In-situ spectroscopic detection of large-scale reorientations of transmembrane α-helices during viroporin 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. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2s transmembrane -helices in-situ during pH-activated channel opening. We quantify this event as a helical tilt from 26{degrees} to 40{degrees} by correlating the experimental results with solid-state NMR-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.

biophysics↗

bioRxiv2021

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 are important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 {beta}-lactamase to TEM-52 towards extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and non-covalent 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. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM {beta}-lactamase antibiotic resistance.

biophysics↗