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bioRxiv2021

Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p-polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.

biophysics↗

bioRxiv2021

Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody

The recently reported "UK variant" of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-[A] resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments. Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

biophysics↗

bioRxiv2021

Modulation of the IKS channel by PIP2 requires two binding sites per monomer

The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. The latter, expressed in the myocardium membrane is complexed with its auxiliary subunits, KCNE1 to generate the so-called IKS current. We present here molecular models of transmembrane domain of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2 lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2 binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2 in KCNE1 modulation of IKS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels.

biophysics↗

bioRxiv2021

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

Worldwide, [~]250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBVs pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex- binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

biophysics↗

bioRxiv2021

Electrophysiological evidence of synergistic auxin transport by interacting Arabidopsis ABCB4 and PIN2 proteins

To understand why both ATP-binding cassette B (ABCB) and PIN-FORMED (PIN) proteins are required for polar auxin transport through tissues, even though only the latter is polarly localized, we biophysically studied their transport characteristics separately and together by whole-cell patch clamping. ABCB4 and PIN2 from Arabidopsis thaliana expressed in human embryonic kidney cells displayed electrogenic activity when CsCl-based electrolytes were used. Current-voltage (I-V) analysis of the activities and modeling the effects of adding the auxin anion (IAA-) as a potential substrate with the Goldman-Hodgkin-Katz equation, demonstrated that ABCB4 and PIN2 were 9-fold and 10-fold more selective for IAA- than Cl-, respectively. Thus, these proteins directly transport IAA-, which was not unequivocally established by previous auxin retention assays. Co-expression of ABCB4 and PIN2 produced an especially significant result. Co-expression synergistically doubled the selectivity for IAA-. An area of two-fold higher selectivity for IAA- that this result indicates will occur in cells with asymmetric PIN2 and symmetric ABCB4 matches what early models found to be necessary to create observed levels of polar auxin transport through tissues. Thus, the requirement for two different proteins appears to be explained by a synergistic effect on selectivity. More substrate details and important pharmacological results are reported.

biophysics↗

bioRxiv2021

PolyQ expansion does not alter the Huntingtin-HAP40 complex

The abnormal amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington disease (HD). At the protein level, this translates into the expansion of a poly-glutamine (polyQ) stretch located at the HTT N-terminus, which renders it aggregation-prone by unknown mechanisms. Here we investigated the effects of polyQ expansion on HTT in a complex with its stabilizing interaction partner huntingtin-associated protein 40 (HAP40). Surprisingly, our comprehensive biophysical, crosslinking mass spectrometry and cryo-EM experiments revealed no major differences in the conformation of HTT-HAP40 complexes of various polyQ length, including 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients) and 128QHTT-HAP40 (extreme polyQ length). Thus, HTT polyQ expansion does not alter the global structure of HTT when associated with HAP40.

biophysics↗

bioRxiv2021

waveRAPID(R) - a robust assay for high-throughput kinetic screens with the Creoptix(R) WAVEsystem

Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as Surface Plasmon Resonance (SPR) or Grating-Coupled Interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process, and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further - up to ten-fold - increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a micro-titer plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique. Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low molecule weight compound screening, and hit-to-lead optimization.

biophysics↗

bioRxiv2021

Elucidating the Mechanical Energy for Cyclization of a DNA Origami Tile

DNA origami has emerged as a versatile method to synthesize nanostructures with high precision. This bottom-up self-assembly approach can produce not only complex static architectures, but also dynamic reconfigurable structures with tunable properties. While DNA origami has been explored increasingly for diverse applications such as biomedical and biophysical tools, related mechanics are also under active investigation. Here we studied the structural properties of DNA origami and investigated the energy needed to deform the DNA structures. We used a single-layer rectangular DNA origami tile as a model system and studied its cyclization process. This origami tile was designed with an inherent twist by placing crossovers every 16 base-pairs (bp), corresponding to a helical pitch of 10.67 bp/turn which is slightly different from that of native B-form DNA (10.5 bp/turn). We used molecular dynamics (MD) simulations based on a coarse-grained model on an open-source computational platform, oxDNA. We calculated the energies needed to overcome the initial curvature and induce mechanical deformation by applying linear spring forces. We found that the initial curvature may be overcome gradually during cyclization and a total of ~33.1 kcal/mol is required to complete the deformation. These results provide insights into the DNA origami mechanics and should be useful for diverse applications such as adaptive reconfiguration and energy absorption.

biophysics↗

bioRxiv2021

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.

biophysics↗

bioRxiv2021

Using a Human Circulation Mathematical Model to Simulate the Effects of Hemodialysis and Therapeutic Hypothermia.

BackgroundThe human blood circulation is an intricate process regulated by multiple biophysical factors. Our patients often suffer from renal disease and atrial fibrillation, and are given treatments such as therapeutic hypothermia, exercise, and hemodialysis. In this work, a hemodynamic mathematical model of human circulation coupled to a representative dialysis machine is developed and used to explore causal mechanisms of our recent clinical observations. MethodsAn ordinary differential equation model consisting of human whole body circulation, baroreflex control, and a hemodialysis machine was implemented. Experimentally informed parameter alterations were used to implement hemodialysis and therapeutic hypothermia. By means of parameter perturbation, four model populations encompassing baseline, dialysed, hypothermia treated, and simultaneous dialysed with hypothermia were generated. In model populations, multiple conditions including atrial fibrillation, exercise, and renal failure were simulated. The effects of all conditions on clinically relevant non-invasive measurables such as heart rate and blood pressure were quantified. A parameter sensitivity analysis was implemented to rank model output influencing parameters in the presented model. ResultsResults were interpreted as alterations of the respective populations mean values and standard deviations of the clinical measurables, both in relation to the baseline population. A clinical measurables smaller standard deviation (in comparison to baseline population) was interpreted as a stronger association between a given clinical measure and the corresponding underlying process, which may permit the use of deducing one by observation of the other. The modelled dialysis was observed to increase systolic blood pressure, vessel shear, and heart rate. Therapeutic hypothermia was observed to reduce blood pressure as well as the intra-population standard deviation (heterogeneity) of blood flow in the large (aorta) and small (kidney) vasculature. Therapeutic hypothermia reduced shear in vessels, suggesting a potential benefit with respect to endothelial dysfunction and maintenance of microcirculatory blood flow. The action of therapeutic hypothermia under conditions such as atrial fibrillation, exercise, and renal failure was to reduce total blood flow, which was applicable in all simulated populations. Therapeutic hypothermia did not affect the dialysis function, but exercise improved the efficacy of dialysis by facilitating water removal. ConclusionsThis study illuminates some mechanisms of action for therapeutic hypothermia. It also suggests clinical measurables that may be used as surrogates to diagnose underlying diseases such as atrial fibrillation.

biophysics↗

bioRxiv2021

An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

Allosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

biophysics↗

bioRxiv2021

Substrate mediated elastic coupling between motile cells modulates inter-cell interactions and enhances cell-cell contact

The mechanical micro-environment of cells and tissues influences key aspects of cell structure and function including cell motility. For proper tissue development, cells need to migrate, interact with other neighbouring cells and form contacts, each of which require the cell to exert physical forces. Cells are known to exert contractile forces on underlying soft substrates. These stresses result in substrate deformation that can affect migratory behavior of cells as well as provide an avenue for cells to sense each other and coordinate their motion. The role of substrate mechanics, particularly its stiffness, in such biological processesis therefore a subject of active investigation. Recent progress in experimental techniques have enabled key insights into pairwise mechanical interactions that control cell motility when they move on compliant soft substrates. Analysis and modeling of such systemsis however still in its nascent stages. Motivated by the role modeling is expected to play in interpreting, informing and guiding experiments, we build a biophysical model for cell migration and cell-cell interactions. Our focus is on situations highly relevant to tissue engineering and regenerative medicine -when substrate traction stresses induced by motile cells enable substrate deformation and serve as a medium of communication. Using a generalizable agent-basedmodel, we compute key metrics of cell motile behavior such as the number of cell-cell contacts over a given time, dispersion of cell trajectories, and probability of permanent cell contact, and analyze how these depend on a cell motility parameter and on substrate stiffness. Our results provide a framework towards modeling the manner in which cells may sense each other mechanically via the substrate and use this information to generate coordinated movements across much longer length scales. Our results also provide a foundation to analyze experiments on the phenomenon known as durotaxis where single cells move preferentially towards regions of high stiffness on patterned substrates.

biophysics↗

bioRxiv2021

Heparin promotes fibrillation of most phenol soluble modulin peptides from S. aureus: a possible strengthening of the bacterial biofilm

Phenol soluble modulins (PSMs) are virulence peptides secreted by different Staphylococcus aureus strains. In addition, PSMs are able to form amyloid fibrils which may strengthen the biofilm matrix. The highly sulfated glycosaminoglycan heparin promotes S.aureus infection but the basis for this is unclear. We hypothesized that heparin promotes PSM fibrillation and in this way aids bacterial colonization. Here we address this hypothesis using a combination of different biophysical techniques along with peptide microarrays. We find that heparin accelerates fibrillation of all -PSMs (except PSM2) and {delta}-toxin, but inhibits {beta}-PSMs fibrillation by blocking nucleation. Given that S. aureus secretes higher levels of -PSMs than {beta}-PSMs peptidess, heparin is likely to overall promote fibrillation. Heparin binding is driven by multiple positively charged lysine residues in -PSMs and {delta}-toxins, whose removal strongly reduces affinity. Binding of heparin does not alter the final fibril conformation. Rather, heparin provides a scaffold to catalyze or inhibit fibrillation. Our findings suggest that heparin may strengthen bacterial biofilm through increased PSM fibrillation. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/434294v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@99c3bdorg.highwire.dtl.DTLVardef@101d9b6org.highwire.dtl.DTLVardef@7c4058org.highwire.dtl.DTLVardef@1647eca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

α-Helical peptidic scaffolds to target α-synuclein pathogenic species with high affinity and selectivity.

-Synuclein aggregation is a key driver of neurodegeneration in Parkinsons disease and related syndromes. Accordingly, obtaining a molecule that targets -synuclein pathogenic assemblies with high affinity and selectivity is a long-pursued objective. Here, we have exploited the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of -helical peptides that bind selectively to these -synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into an unprecedented anti-aggregation potency and the ability to abrogate the oligomers toxicity. With a structure-function relationship in hand, we identified a human peptide expressed in the brain and in the gastrointestinal tract with exceptional binding, antiaggregation, and detoxifying properties, which suggests it might play a protective role against synucleinopathies. The chemical entities we describe here represent a new therapeutic paradigm and are promising tools to assist diagnosis by selectively detecting -synuclein pathogenic species in biofluids.

biophysics↗

bioRxiv2021

Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py

Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation of Fluorescence Accumulation after DNA Damage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present "qFADD.py", an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models. Statement of SignificanceCells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.

biophysics↗

bioRxiv2021

Thermoplasmonic nano-rupture of cells reveals Annexin V function in plasma membrane repair

Maintaining the integrity of the cell plasma membrane (PM) is critical for the survival of cells. While an efficient PM repair machinery can aid survival of healthy cells by preventing influx of extracellular calcium, it can also constitute an obstacle in drug delivery and photothermal therapy. We show how nanoscopic holes can be applied to the cell surface thus allowing identification of molecular components with a pivotal role in PM repair. Cells are punctured by locally heating gold nanostructures at the cell surface which causes nano-ruptures in cellular PMs. Recruitment of annexin V near the hole is found to locally reshape the ruptured plasma membrane. Experiments using model membranes, containing recombinant annexin V, provide further biophysical insight into the ability of annexin V to reshape edges surrounding a membrane hole. The thermoplasmonic method provides a general strategy to monitor the response to nanoscopic injuries to the cell surface.

biophysics↗

bioRxiv2021

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

The translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.

biophysics↗

bioRxiv2021

Consensus tandem tetratricopeptide repeat proteins are complex superhelical nanosprings

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs) -- superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between folded and unfolded conformations. We rationalise the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the centre towards both termini simultaneously. Strikingly, we also directly observe the proteins superhelical tertiary structure in the force signal. Using protein engineering, crystallography and single-molecule experiments, we show how the superhelical geometry can be altered by carefully placed amino-acid substitutions and examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications. Significance statementRepetition of biological building blocks is crucial to modulating and diversifying structure and function of biomolecules across all organisms. In tandem-repeat proteins, the linear arrangement of small structural motifs leads to the formation of striking supramolecular shapes. Using a combination of single-molecule biophysical techniques and modelling approaches, we dissect the spring-like nature of a designed repeat protein and demonstrate how its shape and mechanics can be manipulated by design. These novel insights into the biomechanical and biochemical characteristics of this protein class give us a methodological basis from which to understand the biological functions of repeat proteins and to exploit them in nanotechnology and biomedicine.

biophysics↗