Biology subjects

Boxer, S. G.

Publications and source records attributed to Boxer, S. G..

7 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↗

bioRxiv2025

Magnetic resonance control of reaction yields through genetically-encoded protein:flavin spin-correlated radicals in a live animal

Radio-frequency (RF) magnetic fields can influence reactions involving spin-correlated radical pairs. This provides a mechanism by which RF fields can influence living systems at the biomolecular level. Here we report the modification of the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, induced by a combination of static and RF magnetic fields. Resonance features in the protein fluorescence intensity were observed near the electron spin resonance frequency at the corresponding static magnetic field strength. This effect was measured at room temperature both in vitro and in the nematode C. elegans, genetically modified to express the RFP mScarlet. These observations suggest that the magnetic field effects measured in RFP-flavin systems are due to quantum-correlated radical pairs. Our experiments demonstrate that RF magnetic fields can influence dynamics of reactions involving RFPs in biologically relevant conditions, and even within a living animal. These results have implications for the development of a new class of genetic tools based on RF manipulation of genetically-encoded quantum systems.

biophysics↗

bioRxiv2022

Membrane localization accelerates association under conditions relevant to cellular signaling

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here we directly compare an identical association reaction in solution and on supported membranes. The measured rate constants show that for 10 m-radius spherical cell, association is 15-25-fold faster at the membrane than in the cytoplasm. The advantage is cell size-dependent, and for typical ~1 m prokaryotic cells it should be essentially negligible. Rate enhancement is attributable to a combination of closer proximity of the signaling molecule to its targets after translocation and the higher efficiency of a two-dimensional search.

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↗

bioRxiv2021

Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses. Statement of SignificanceTo replicate, enveloped viruses, like influenza A virus, must successfully deliver their contents to a host cell through viral membrane fusion. Most single-virus fusion assays require extensive fluorescent labeling of virions which can be perturbative to fusion kinetics. Here, we utilize content-labeled vesicles in a single-virus content mixing assay, which eliminates the need to fluorescently label virus contents. We use this assay to show that target membrane cholesterol increases the fraction of stable influenza virus content mixing events. This assay also enables the study of target membrane destabilization due to viral fusion peptide engagement.

biophysics↗

bioRxiv2019

Target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including: initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a new fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles, and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, while the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature. Statement of SignificanceInfluenza A virus is responsible for millions of cases of flu each year. In order to replicate, influenza must enter a host cell through virus membrane fusion, and cholesterol in the target membrane is vital to the dynamics of this process. We report a receptor-free, single virus fusion assay that requires no fluorescent labeling of virus particles. We use this assay to show that cholesterol increases the fraction of fusion events in a manner that is correlated with the spontaneous curvature of the target membrane but is independent of receptor binding. This assay represents a promising strategy for studying viral fusion processes of other enveloped viruses.

biophysics↗

bioRxiv2019

Detecting and controlling dye effects in single-virus fusion experiments

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.\n\nStatement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

biophysics↗