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bioRxiv2021

Single-molecule biophysics experiments in silico: Towards a physical model of a replisome

The interpretation of single-molecule experiments is frequently aided by computational modeling of biomolecular dynamics. The growth of computing power and ongoing validation of computational models suggest that it soon may be possible to replace some experiments out-right with computational mimics. Here we offer a blueprint for performing single-molecule studies in silico using a DNA binding protein as a test bed. We demonstrate how atomistic simulations, typically limited to sub-millisecond durations and zeptoliter volumes, can guide development of a coarse-grained model for use in simulations that mimic experimental assays. We show that, after initially correcting excess attraction between the DNA and protein, qualitative consistency between several experiments and their computational equivalents is achieved, while additionally providing a detailed portrait of the underlying mechanics. Finally the model is used to simulate the trombone loop of a replication fork, a large complex of proteins and DNA.

biophysics↗

bioRxiv2022

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD-simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

biophysics↗

bioRxiv2022

Early selection of the amino acid alphabet was adaptively shaped by biophysical constraints of foldability

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 early AAs, and that the 10 late AAs were products of biosynthetic pathways. However, many non-proteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet, and Would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluated the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. We show that foldability was a critical factor in the selection of the canonical alphabet. Unbranched aliphatic and short-chain basic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are over-solubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides secondary structure potential. Our results support the view that despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of the alphabet evolution.

biophysics↗

bioRxiv2022

Biophysical analysis of Gaussia Luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine.

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but Glucs fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine, a non-oxidizable coelenterazine analog, as a probe to investigate its binding mode to GLuc. Interestingly, the biochemical studies of GLuc inhibition by azacoelenterazine also led us to find that salt, and monovalent anions, are required for GLucs bioluminescence, which seems reasonable for a sea-dwelling creature. The NMR-based investigation, using chemical shift perturbations monitored by 15NH-HSQC, suggested that CTZ binds to residue in or near the hydrophobic cavity. Of note is that these NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.

biophysics↗

bioRxiv2023

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements severely limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) Inhibitor-induced changes of traction forces and fiber alignment and density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

biophysics↗

bioRxiv2023

Biophysical modeling identifies an optimal hybrid amoeboid-mesenchymal phenotype for maximal T cell migration speeds

Despite recent advances in understanding cell migration mechanics, the principles governing rapid T cell movement remain unclear. Efficient migration is critical for antitumoral T cells to locate and eliminate cancer cells. To investigate the upper limits of cell speed, we developed a hybrid stochastic-mean field model of bleb-based cell motility. Our model suggests that cell-matrix adhesion-free bleb-based migration is highly inefficient, challenging the feasibility of cell swimming/adhesion-independent migration as a primary fast motility mode. Instead, we show that T cells can achieve rapid migration by combining bleb formation with adhesion-based forces. Supporting our predictions, our three-dimensional gel experiments confirm that T cells migrate significantly faster under adherent conditions than in adhesion-free environments. These findings highlight the mechanical constraints of T cell motility and suggest that modifying tissue adhesion properties in a controlled manner could enhance immune cell infiltration into tumors. Our computational and experimental work provides insights for optimizing T cell-based immunotherapies. While antifibrotic treatments could alter the tumor microenvironment, indiscriminate reduction of adhesion may not be ideal for T cell infiltration and motility, highlighting the need for targeted antifibrotic strategies.

biophysics↗

bioRxiv2024

The interplay between proton diffusion across biological membranes and their biophysical properties highlights the role of defects in mixed lipid membranes

Proton circuits within biological membranes are at the heart of natural bioenergetic systems, whereas different biological membranes are characterized by different lipid compositions. In this study, we investigate how the composition of mixed lipid membranes influences the proton transfer (PT) properties of the membrane by following the excited-state PT (ESPT) process from a tethered probe to the membrane with time-scales and length-scales of PT that are relevant to bioenergetic systems. Two processes can happen during ESPT: the initial PT from the probe to the membrane at short timescales, followed by diffusion of dissociated protons around the probe on the membrane, and the possible geminate recombination with the probe at longer timescales. Here, we use membranes that are composed of mixtures of phosphatidylcholine (PC) and phosphatidic acid (PA). We show that the changes in the ESPT properties are not monotonous with the concentration of the lipid mixture; at low concentration of PA in PC, we find that the membrane is a poor proton acceptor. Molecular dynamics simulations indicate that at this certain lipid mixture, the membrane has the least defects (more structured and unflawed). Accordingly, we suggest that defects can be an important factor in facilitating PT. We further show that the composition of the membrane affects the geminate proton diffusion around the probe, whereas, on a time-scale of tens of nanoseconds, the dissociated proton is mostly lateral restricted to the membrane plane in PA membranes, while in PC, the diffusion is less restricted by the membrane.

biophysics↗

bioRxiv2024

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.

Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being successfully trafficked to the cell membrane and capable of binding PIP2 (phosphatidylinositol-4,5- bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K+ current, albeit through different molecular mechanisms. Co-expression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K+ flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.

biophysics↗

bioRxiv2024

Biophysical Fluid Dynamics in a Petri Dish

The humble Petri dish is perhaps the simplest setting in which to examine the locomotion of swimming organisms, particularly those whose body size is tens of microns to millimetres. The fluid layer in such a container has a bottom no-slip surface and a stress-free upper boundary. It is of fundamental interest to understand the flow fields produced by the elementary and composite singularities of Stokes flow in this geometry. Building on the few particular cases that have previously been considered in the literature, we study here the image systems for the primary singularities of Stokes flow subject to such boundary conditions --the stokeslet, rotlet, source, rotlet dipole, source dipole and stresslet --paying particular attention to the far-field behavior. In several key situations, the depth-averaged fluid flow is accurately captured by the solution of an associated Brinkman equation whose screening length is proportional to the depth of the fluid layer. The case of hydrodynamic bound states formed by spinning microswimmers near a no-slip surface, discovered first using the alga Volvox, is reconsidered in the geometry of a Petri dish, where the powerlaw attractive interaction between microswimmers acquires unusual exponentially screened oscillations.

biophysics↗

bioRxiv2024

Biophysics of microRNA-34a targeting and its influence on down-regulation

microRNAs (miRNAs) regulate target mRNA expression post-transcriptionally through their association with Argonaute 2 (AGO2) proteins. Predicting the efficiency of mRNA repression by miRNA has been limited by our comprehension of the structure-function relationship within miRNA binding sites. Using a combination of EMSA, luciferase reporter assays, and structural probing, we investigated the interaction between the human tumour suppressor miR-34a and 12 mRNA targets. Comparison of direct RNA:RNA interactions and those within the functional AGO2 protein revealed that the isolated mRNA:miRNA duplex serves as a strong predictor for duplex affinity and structure within AGO2. Our findings reveal that AGO2 has a bidirectional capacity to modulate affinity; weakening tight RNA:RNA binders while strengthening weak ones. We identified three distinct structural groups that form upon miR-34a binding and reveal a novel structural group that exhibits a guide strand bulge. MD simulations indicate a conceivable fit of this miRNA-bulge structure within AGO2. Our results demonstrate that the structural characteristics of mRNA:miRNA duplexes could serve as contributing determinants of repression efficacy.

biophysics↗

bioRxiv2024

Biophysical metabolic modeling of complex bacterial colony morphology

Microbial colony growth is shaped by the physics of biomass propagation and nutrient diffusion, and by the metabolic reactions that organisms activate as a function of the surrounding environment. While microbial colonies have been explored using minimal models of growth and motility, full integration of biomass propagation and metabolism is still lacking. Here, building upon our framework for Computation of Microbial Ecosystems in Time and Space (COMETS), we combine dynamic flux balance modeling of metabolism with collective biomass propagation and demographic fluctuations to provide nuanced simulations of E. coli colonies. Simulations produced realistic colony morphology, consistent with our experiments. They characterize the transition between smooth and furcated colonies and the decay of genetic diversity. Furthermore, we demonstrate that under certain conditions, biomass can accumulate along "metabolic rings" that are reminiscent of coffee-stain rings, but have a completely different origin. Our approach is a key step towards predictive microbial ecosystems modeling.

biophysics↗

bioRxiv2024

Biophysical mapping of TREM2-ligand interactions reveals shared surfaces for engagement of multiple AD ligands

TREM2 is a signaling receptor expressed on microglia that has emerged as an important potential drug target for Alzheimers disease and other neurodegenerative diseases. While a number of TREM2 signaling ligands have been identified, little is known regarding the structural details of how it engages them. To better understand this, we created a protein library of 28 different TREM2 variants and 11 different sTREM2 variants that could be used to map interactions with various ligands using biolayer interferometry (BLI). The variants are located in previously identified putative binding surfaces on TREM2 called the hydrophobic site, basic site, and site 2. We found that mutations to the hydrophobic site ablated binding to apoE4, oAb42, and TDP-43. Competition binding experiments further supported that apoE4 and oAb42 share overlapping binding sites on TREM2. In contrast, binding to IL-34 was mediated by the basic site at a surface centering on R76. Competition binding experiments validated a unique site for IL-34, showing little to no competition with either oAb42 or apoE4. Altogether, our results suggest that TREM2 utilizes the hydrophobic site (consisting of CDR1, CDR2, and CDR3) as a common site to engage multiple ligands, and further implies that pharmaceutical strategies targeting this surface might be effective to modulate TREM2 functions.

biophysics↗

bioRxiv2024

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2{beta} ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

biophysics↗

bioRxiv2024

EGFR meets hATG8s - Biophysical and structural insights supporting a unique role of GABARAP during receptor trafficking

The human ATG8 family member GABARAP is involved in numerous autophagy-related and - unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances EGFR degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within the EGFR, the first of which (LIR1) is selected as GABARAP binding site in-silico. Indeed, in-vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting a canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

biophysics↗

bioRxiv2024

Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naive cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

biophysics↗

bioRxiv2024

Reliable reconstruction of cricket song from biophysical models and preserved specimens

Predicting the function of a biological structure solely from its morphology can be a very powerful tool in several fields of biology, but especially in evolutionary reconstruction. In the field of invertebrate acoustic communication, reconstructing the acoustic properties of sound-producing forewings in crickets has been based on two very divergent methods, finite element modelling (FEM) and vibrometric measurements from preserved specimens. Both methods, however, make strong simplifying assumptions which have not been tested and the reliability of inferences made from either method remains in question. Here we rigorously test and refine both reconstruction methods using the well-known Teleogryllus oceanicus model system and determine the appropriate conditions required to reconstruct the vibroacoustic behaviour of male forewings. We find that when using FEM it is not necessary to assume simplified boundary conditions if the appropriate parameters are found. When using preserved specimens, we find that the sample needs to be rehydrated for reliable reconstruction, however, it may be possible to accomplish rehydration in silico using FEM. Our findings provide a refined methodology for the reliable reconstruction of cricket songs, whether from fossils or preserved specimens from museums or field collections.

biophysics↗

bioRxiv2024

Modulation of Host Cell Membrane Biophysics Dynamics by Neospora caninum: A Study Using LAURDAN Fluorescence with Hyperspectral Imaging and Phasor Analysis

Neospora caninum is known to manipulate host cell organelles and recruit lipids for its survival. However, the impact of this lipid redistribution on host cell membranes remains poorly understood. This study used LAURDAN fluorescence, hyperspectral imaging, and phasor plot analysis to investigate how N. caninum modifies membrane order in Vero cells. The results revealed a significant decrease in host cell plasma and internal membrane order upon infection, suggesting that cholesterol is redistributed from the host plasma membrane to the parasitophorous vacuoles. To mimic cholesterol depletion, uninfected cells were treated with methyl-{beta}-cyclodextrin (MBCD), which increased membrane fluidity. Conversely, replenishing infected cells with cholesterol-loaded MBCD restored membrane fluidity to levels lower than control cells, indicating cholesterol enrichment. These findings provide novel insights into how N. caninum modulates host cell membrane dynamics through lipid manipulation, potentially aiding its intracellular survival.

biophysics↗

bioRxiv2024

Biophysical Basis of Paracellular Barrier Modulation by a Pan-Claudin-Binding Molecule

Claudins are a 27-member protein family that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport across tissues by forming molecular barriers between cells. Claudin-binding molecules thus hold promise for modulating tight junction permeability to deliver drugs or as therapeutics to treat tight junction-linked disease. The development of claudin-binding molecules, however, is hindered by their intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) we developed binds directly to 10 claudin subtypes with nanomolar affinity by targeting claudins paracellular-exposed surface. Application of this sFab to cells that model intestinal epithelium show that it opens the paracellular barrier comparable to a known, but application limited, tight junction modulator. This novel pan-claudin-binding molecule can probe claudin or tight junction structure and holds potential as a broad modulator of tight junction permeability for basic or translational applications.

biophysics↗