Biology subjects

Quinn, S. D.

Publications and source records attributed to Quinn, S. D..

14 recordsLinked to original sources

bioRxiv2025

Detergent-Triggered Membrane Remodelling Monitored via Intramembrane Fluorescence De-Quenching

Detergent-induced membrane solubilization is important for several biotechnological applications including membrane protein isolation, cell lysis and virus inactivation. The thermodynamic details of the underlying process have been previously examined, but the mechanistic details remain largely underexplored owing in part, to a lack of suitable technologies capable of assessing nanoscopic membrane disruption events. Key open questions include: how do detergents remodel the membrane structure at sub-solubilizing concentrations? And what is the sequence of morphological transitions that lead up to solubilization? Here, we introduce a single-colour assay based on the fluorescence de-quenching of membrane-integrated fluorophores as a sensitive and generalizable tool to probe nanoscale membrane remodelling events induced by detergents. We demonstrate, using fluorescence spectroscopy and time-correlated single photon counting, that the widely used detergent Triton X-100 triggers substantial morphological changes at concentrations below its critical micellar concentration. Moreover, by taking advantage of single vesicle fluorescence lifetime imaging and scanning electron microscopy, we reveal that the swelling step involves a morphological transition from spherical vesicles to toroidal structures, providing direct evidence for detergent-driven membrane reorganization prior to solubilization. Our findings support and refine a multistep model of detergent-induced membrane solubilization, positioning fluorescence de-quenching as a tool for detecting conformational intermediates. We show that the fluorescence de-quenching approach performs robustly across multiple cyanine-based probes and experimental conditions and its nanoscale sensitivity provides a platform from which to interrogate membrane perturbations induced by a wide variety of molecular disruptors, including those with important biomedical significance.

biophysics↗

bioRxiv2025

A DNA scaffold approach facilitates 5' labelling of the SARS-CoV-2 RNA pseudoknot for smFRET investigation

Single-molecule Forster resonance energy transfer (smFRET) studies of highly structured RNA molecules are often frustrated by issues with efficient dye conjugation. Here, we develop a DNA scaffold-based labelling strategy, and apply it to the frameshift-stimulating RNA pseudoknot from the SARS-CoV-2 genome. We prepare FRET-active reporters containing both Cy3 (donor) and Cy5 (acceptor) molecules and conduct measurements on freely diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. We identify that freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl-dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X-ray and cryo-EM structures. This work is a proof-of-principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches towards the study of RNA conformational dynamics in pseudoknots, riboswitches and other structured RNA elements.

biophysics↗

bioRxiv2025

Electronic Actuation of Surface-Immobilized, pH-Responsive DNA Nanoswitches

Dynamic DNA machines exploit the specificity of base pairing and/or sensitivity to the local environment to control the reversible switching of DNA constructs between conformational states. One such example are pH-sensitive DNA nanoswitches that can be actuated by proton-mediated Hoogsteen interactions within a DNA triplex domain. To date, studies of pH-sensitive DNA nanoswitches have largely focused on DNA machines that are freely diffusing in the solution phase. For many applications, it is advantageous to integrate these dynamic DNA machines with solid-state devices, requiring immobilization on surfaces. Here, we explore the switching of a pH-sensitive DNA triplex immobilized on a surface as a dense, 2-dimensional DNA monolayer. DNA nanoswitches were assembled onto surfaces via thiol chemistry and pH-controlled conformational switching of the constructs examined using quartz crystal microbalance with dissipation monitoring (QCM-D). These QCM-D experiments indicate that despite the high density of DNA within the monolayer (1012 molecules/cm2), pH-switching between open and closed states is retained following immobilization. Moreover, conformational switching of DNA constructs within the monolayer remains highly reversible and repeatable, with negligible reduction in switching efficiency observed over 20 switching cycles. DNA switching experiments were also performed in the solution phase using single-molecular Forster resonance energy transfer (smFRET) and circular dichroism (CD) techniques to confirm their pH responsitivity. Finally, we demonstrate electrically driven, localised, and addressable switching of the DNA triplex by employing electrochemical reduction and oxidation of water at an electrode surface, further demonstrating the potential of the technology for surface-immobilized dynamic DNA machines. This study not only provides insight into the actuation of DNA machines on-surface but also supports the development of new technologies such as hybrid electronic-DNA technologies able to store and process information using both molecular and electronic inputs.

biophysics↗

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

bioRxiv2023

Multiple Intermediates in the Detergent-Induced Fusion of Lipid Vesicles

Detergent-induced vesicle interactions, critical for applications including virus inactivation, varies according to the detergent type and membrane composition, but the underlying mechanistic details remain underexplored. Here, we use a lipid mixing assay based on Forster resonance energy transfer (FRET), and single-vesicle characterization approaches to identify that sub-micron sized vesicles are induced to fuse by the non-ionic detergent Triton-X 100. We demonstrate that the process is a multi-step mechanism, characterized by discrete values of FRET efficiency between membrane-embedded fluorophores, and involves permeabilization, vesicle docking, hemi-fusion and full lipid mixing at sub-solubilizing detergent concentrations. We also dissect the kinetics of vesicle fusion to surface-tethered vesicles using a label-free quartz-crystal microbalance with dissipation monitoring approach, opening a platform for biotechnology applications. The presented strategies provide mechanistic insight into the dynamics of vesicle fusion and have implications for applications including drug delivery and sensor development where transport and manipulation of encapsulated cargo is essential.

biophysics↗

bioRxiv2022

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

In eukaryotes, intracellular physicochemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion, and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalised in cells is tracked and physicochemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Forster Resonance Energy Transfer (FRET) biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multidimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterise in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells, and discuss limitations of this approach including impairment of cellular function.

biophysics↗

bioRxiv2022

Sticker-and-Linker Model for Amyloid BetaCondensation and Fibrillation

A major pathogenic hallmark of Alzheimers disease is the presence of neurotoxic plaques composed of amyloid beta (A{beta}) peptides in patients brains. The pathway of plaque formation remains elusive, though some clues appear to lie in the dominant presence of A{beta}1-42 in these plaques despite A{beta}1-4 making up approximately 90% of the A{beta} pool. We hypothesise that this asymmetry is driven by the hydrophobicity of the two extra amino acids that are incorporated in A{beta}1-42. To investigate this hypothesis at the level of single molecules, we have developed a molecular sticker-and-linker lattice model of unfolded A{beta}. The model protein has a single sticker that may reversibly dimerise and elongate into semi-flexible linear oligomers. The growth is hampered by excluded-volume interactions that are encoded by the hydrophilic linkers but is rendered cooperative by the attractive interactions of hydrophobic linkers. For sufficiently strong hydrophobicity, the chains undergo liquid-liquid phase-separation (LLPS) into condensates that facilitate the nucleation of fibres. We find that a small fraction of A{beta}1-40 in a mixture of A{beta}1-40 and A{beta}1-42 shifts the critical concentration for LLPS to lower values. This study provides theoretical support for the hypothesis that LLPS condensates act as a precursors for aggregation and provides an explanation for the A{beta}1-42-enrichment of aggregates in terms of hydrophobic interactions.

neuroscience↗

bioRxiv2022

Crowding induced morphological changes in synthetic lipid vesicles determined using smFRET

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely-diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

biophysics↗

bioRxiv2022

Tween-20 induces the structural remodelling of single lipid vesicles

The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

biophysics↗

bioRxiv2021

Ligand-induced transmembrane conformational coupling in monomeric EGFR

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. While receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution from the single helix of a monomeric receptor has been challenging to isolate due to the complexity and ligand-dependence of the receptor-receptor interactions. By combining membrane nanodiscs produced wtih cell-free expression, single-molecule Forster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes are common to single-pass membrane proteins.

biophysics↗

bioRxiv2020

Single-molecule FRET dynamics of molecular motors in an ABEL Trap

Single-molecule Forster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL traps inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

biophysics↗

bioRxiv2020

The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotech-nological applications, including viral inactivation and protein extraction. Although the ensemble thermo-dynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multi-angle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single sub-micron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on in-tact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization and we expect the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

biophysics↗

bioRxiv2019

Unveiling the multi-step solubilization mechanism of single vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Forster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

biophysics↗