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bioRxiv2023

Sculpting DNA-based synthetic cells through phase separation and phase-targeted activity

Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localised. Inspired by membrane-less compartmentalisation in biological cells, here we demonstrate how micro-phase separation can be used to engineer heterogeneous cell-like architectures with programmable morphology and compartment-targeted activity. The synthetic cells selfassemble from amphiphilic DNA nanostructures, producing core-shell condensates due to size-induced de-mixing. Lipid deposition and phase-selective etching are then used to generate a porous pseudo-membrane, a cytoplasm analogue, and membrane-less organelles. The synthetic cells can sustain RNA synthesis via in vitro transcription, leading to cytoplasm and pseudo-membrane expansion caused by an accumulation of the transcript. Our approach exemplifies how architectural and functional complexity can emerge from a limited number of distinct building blocks, if molecular-scale programmability, emergent biophysical phenomena, and biochemical activity are coupled to mimic those observed in live cells.

biophysics↗

bioRxiv2023

Mechanism of tension propagation in cell membranes

The propagation of the membrane tension perturbations is a, potentially, essential mechanism of the mechanical signal transduction along surfaces of live cells. The efficiency of this process is determined by the propagation speed, which turned to be a hot and a controversial topic of the Cell Biophysics. In a stark contrast to the earlier results and expectations, the recent studies in several cell types revealed a wide range of the tension propagation speeds beginning from the strikingly low ones challenging the significance of the process and up to relatively high biologically relevant rates. The previously suggested models of the tension propagation have been based on assuming an unrealistic softness of the membranes for the stretching-compression deformations, which challenges the model ability to account for the observations. Here, we consider a different physics of the generation and the propagation of tension perturbations in cell membranes. We propose the tension to be controlled by an intra-cellular pressure and the propagation of the tension perturbations to be mediated by a membrane area redistribution between compartments, to which cell membranes are divided by the proteinic barriers, according to the picket-fence model. Using the established elastic features of cell membranes including their effective non-stretchability, this mechanism quantitatively accounts for the slowness of the propagation process and gives a natural explanation of the wide range of the observed propagation speeds. The model predictions are amenable to a direct experimental verification by controlled osmotic pressure variations.

biophysics↗

bioRxiv2023

An easy-to-use microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding

Conventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellular assemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they are often hard to use and do not allow the control of compressive forces. Here, we present a set of microfluidic devices which all rely on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable sealed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat is an easy-to-use microfluidic device that allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression.

biophysics↗

bioRxiv2023

Characterization of uranyl ion binding to amyloid beta (Aβ) peptides: effects on Aβ structure and aggregation

AbstractUranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions including Alzheimers disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is deposition of amyloid plaques, consisting mainly of amyloid-{beta} (A{beta}) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric A{beta} aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase A{beta} production, and these metals bind to A{beta} peptides and modulate their aggregation. Possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between A{beta} peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to A{beta} peptides with affinities in the micromolar range, induce structural changes in A{beta} monomers and oligomers, and inhibit A{beta} fibrillization. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

biophysics↗

bioRxiv2023

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

biophysics↗

bioRxiv2023

Novel structural insights on full-length human RAD52: Cryo-EM and beyond

Human RAD52 is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer synthetic lethality strategies since its inhibition or modulation, under specific genetic conditions, was proved to enhance therapies efficacy in various cancer cell types. Although the interest in RAD52 has exponentially grown in the last decade, most information about its structure and mechanism of action is still missing. This work provides novel insights into full-length RAD52 (RAD52 FL) protein, focusing on its structural and functional characterization. The Cryo-Electron Microscopy (Cryo-EM) structure of RAD52 FL, here presented at a resolution (2.16 [A]) higher than the one currently available for RAD52 N-terminal X-ray structure, allows hypothesizing the role of individual amino acid residues. While the N-terminal region of RAD52 FL is structured in an undecameric ring, the C-terminal part is intrinsically disordered as fully characterized through SAXS and biophysical analyses. These detailed (atomic level) structural analyses will substantially impact future characterizations of RAD52 mechanisms of action and inhibitors development, particularly in the context of novel approaches to synthetic lethality.

biophysics↗

bioRxiv2023

Allosteric coupling asymmetry mediates paradoxical activation of BRAF

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active C-in state. This provides a structural mechanism for how binding of only a single C-in inhibitor molecule can induce potent BRAF dimerization and activation.

biophysics↗

bioRxiv2023

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene (DYSF). This disease has two known pathogenic missense mutations that occur within dysferlins C2A domain, namely C2AW52R and C2AV67D. Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlins ability to repair cells. The failure of C2AW52R and C2AV67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes {beta}-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1{beta}. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2AW52R and C2AV67D, as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

biophysics↗

bioRxiv2023

Sizes, Conformational fluctuations, and SAXS profiles for Intrinsically Disordered Proteins

The preponderance of Intrinsically Disordered Proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, folded proteins, RNA, and DNA for functional purposes, have made it important to quantitatively characterize their biophysical properties. Toward this end, we developed the transferable Self-Organized Polymer (SOP-IDP) model to calculate the properties of several IDPs. The values of the radius of gyration (Rg) obtained from SOP-IDP simulations are in excellent agreement (correlation coefficient of 0.96) with those estimated from SAXS experiments. For AP180 and Epsin, the predicted values of the hydrodynamic radii (Rhs) are in quantitative agreement with those from Fluorescence Correlation Spectroscopy (FCS) experiments. Strikingly, the calculated SAXS spectra for thirty-six IDPs are also nearly superimposable on the experimental profiles. The dependence of Rg and the mean end-to-end distance (Ree) on chain length, N, follows Florys scaling law, R {approx} aN 0.588 ( = g, and e), suggesting that globally IDPs behave as synthetic polymers in a good solvent. The values of ag, and ae are 0.20 nm and 0.48 nm respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, are negligible for Rg and Ree. In contrast, only by accounting for the finite sizes of the IDPs, the dependence of experimentally measurable Rh on N can be quantitatively explained using{nu} = 0.588. Although Flory scaling law captures the estimates for Rg, Ree, and Rh accurately, the spread of the simulated data around the theoretical curve is suggestive of of sequence-specific features that emerge through a fine-grained analysis of the conformational ensembles using hierarchical clustering. Typically, the ensemble of conformations partitiones into three distinct clusters, having different equilibrium populations and structural properties. Without any further readjustments to the parameters of the SOP-IDP model, we also obtained excellent agreement with paramagnetic relaxation enhancement (PRE) measurements for -synuclein. The transferable SOP-IDP model sets the stage for several applications, including the study of phase separation in IDPs and interactions with nucleic acids.

biophysics↗

bioRxiv2023

Can motional dynamics account for the cytotoxicity of beta amyloid oligomers?

The underlying biophysical principle governing the cytotoxicity of the oligomeric aggregates of {beta}-amyloid (A{beta}) peptides has long been an enigma. Here we show that the size of A{beta}40 oligomers can be actively controlled by incubating the peptides in reverse micelles. Our approach allowed for the first time a detailed comparison of the structures and dynamics of two A{beta}40 oligomers of different size, viz., 10 and 23 nm, by solid-state NMR. From the chemical shift data, we infer that the conformation of the residues from K16 to K28 are different between the 10-nm and 23-nm oligomers. We find that the 10-nm oligomers are more cytotoxic, and the molecular motions of their charged residues are more dynamic. Interestingly, the residue A21 exhibits an unusually high structural rigidity. Our data raise the interesting possibility that the cytotoxicity of A{beta}40 oligomers could also be correlated to the motional dynamics of the charged residues.

biophysics↗

bioRxiv2023

Direct prediction of intrinsically disordered protein conformational properties from sequence

Intrinsically disordered regions (IDRs) are ubiquitous across all domains of life and play a range of functional roles. While folded domains are generally well-described by a single 3D structure, IDRs exist in a collection of interconverting states known as an ensemble. This structural heterogeneity means IDRs are largely absent from the PDB, contributing to a lack of computational approaches to predict ensemble conformational properties from sequence. Here we combine rational sequence design, large-scale molecular simulations, and deep learning to develop ALBATROSS, a deep learning model for predicting IDR ensemble dimensions from sequence. ALBATROSS enables the instantaneous prediction of ensemble average properties at proteome-wide scale. ALBATROSS is lightweight, easy-to-use, and accessible as both a locally installable software package and a point-and-click style interface in the cloud. We first demonstrate the applicability of our predictors by examining the generalizability of sequence-ensemble relationships in IDRs. Then, we leverage the high-throughput nature of ALBATROSS to characterize emergent biophysical behavior of IDRs within and between proteomes. Update from previous versionO_LIThis preprint reports an updated version of the ALBATROSS network weights trained on simulations of over 42,000 sequences. C_LIO_LIIn addition, we provide new colab notebooks that enable proteome-wide IDR prediction and annotation in minutes. C_LIO_LIAll conclusions and observations made in versions 1 and 2 of this manuscript remain true and robust. C_LI

biophysics↗

bioRxiv2023

De novo amyloid peptides with subtle sequence variations differ in their self-assembly and nanomechanical properties

Proteinaceous amyloids are well known for their widespread pathological roles but lately have emerged also as key components in several biological functions. The remarkable ability of amyloid fibers to form tightly packed conformations in a cross {beta}-sheet arrangement manifests in their robust enzymatic and structural stabilities. These characteristics of amyloids make them attractive for designing proteinaceous biomaterials for various biomedical and pharmaceutical applications. In order to design customizable and tunable amyloid nanomaterials, it is imperative to understand the sensitivity of the peptide sequence for subtle changes based on amino acid position and chemistry. Here we report our results from four rationally-designed amyloidogenic decapeptides that subtly differ in hydrophobicity and polarity at positions 5 and 6. We show that making the two positions hydrophobic renders the peptide with enhanced aggregation and material properties while the introduction of polar residues in position 5 dramatically changes the structure and nanomechanical properties of the fibrils formed. A charged residue at position 6, however, completely abrogates amyloid formation. In sum, we show that subtle changes in the sequence do not make the peptide innocuous but rather sensitive to aggregation, reflected in the biophysical and nanomechanical properties of the fibrils. We conclude that tolerance of peptide amyloid for subtle changes in the sequence should not be neglected for the effective design of customizable amyloid nanomaterials.

biophysics↗

bioRxiv2023

Human hnRNPA1 reorganizes telomere-bound Replication Protein A

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence SummaryAt the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

biophysics↗

bioRxiv2023

Quantitative Models of Molecular Dynamics from Sparse Simulation and Experimental Data

Long-timescale behavior of proteins is fundamental to many biological processes. Molecular Dynamics (MD) simulations and biophysical experiments are often used to study protein dynamics. However, high computational demands of MD limit what timescales are feasible to study, often missing rare events, which are critical to explain experiments. On the other hand, experiments are limited by low resolution. We present dynamic Augmented Markov models (dynAMMo) to bridge the gap between these data and overcome their respective limitations. For the first time, dynAMMo enables the construction of mechanistic models of slow exchange processes that have been not observed in MD data by integrating dynamic experimental observables. As a consequence, dynAMMo allows us to bypass costly and extensive simulations, yet providing mechanistic insights of the system. Validated with controlled model systems and a well-studied protein, dynAMMo offers a new approach to quantitatively model protein dynamics on long timescales in an unprecedented manner.

biophysics↗

bioRxiv2023

Bilayer tension-induced clustering of the UPR sensor IRE1

The endoplasmic reticulum acts as a protein quality control center where a range of chaperones and foldases facilitates protein folding. IRE1 is a sensory trans-membrane protein that transduces signals of proteotoxic stress by forming clusters and activating a cellular program called the unfolded protein response (UPR). Recently, membrane thickness variation due to membrane compositional changes have been shown to drive IRE1 cluster formation, activating the UPR even in the absence of proteotoxic stress. Here, we demonstrate a direct relationship between bilayer tension and UPR activation based on IRE1 dimer stability. The stability of the IRE1 dimer in a (50%DOPC-50%POPC) membrane at different applied bilayer tensions was analyzed via molecular dynamics simulations. The potential of mean force for IRE1 dimerization predicts a higher concentration of IRE1 dimers for both tensed and compressed ER membranes. This study shows that IRE1 may be a mechanosensitive membrane protein and establishes a direct biophysical relationship between bilayer tension and UPR activation. HighlightsO_LIMechanical perturbation of the ER membrane favor oligomerization. C_LIO_LIBoth tension and compression promote IRE1 dimer formation. C_LIO_LIIRE1 is mechanosensitive, potentially the UPR to changes in ER membrane tension and compression. C_LI

biophysics↗

bioRxiv2023

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it an interesting player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. The possible application of these microreactors in bioremediation is discussed.

biophysics↗

bioRxiv2023

Effective cell membrane tension protects red blood cells against malaria invasion

A critical step in how malaria parasites invade red blood cells (RBCs) is the wrapping of the membrane around the egg-shaped merozoites. Recent experiments have revealed that RBCs can be protected from malaria invasion by high membrane tension. While cellular and biochemical aspects of parasite actomyosin motor forces during the malaria invasion have been well studied, the important role of the biophysical forces induced by the RBC membrane-cytoskeleton composite has not yet been fully understood. In this study, we use a theoretical model for lipid bilayer mechanics, cytoskeleton deformation, and membrane-merozoite interactions to systematically investigate the influence of effective RBC membrane tension, which includes contributions from the lipid bilayer tension, spontaneous tension, interfacial tension, and the resistance of cytoskeleton against shear deformation on the progression of membrane wrapping during the process of malaria invasion. Our model reveals that this effective membrane tension creates a wrapping energy barrier for a complete merozoite entry. We calculate the tension threshold required to impede the malaria invasion. We find that the tension threshold is a nonmonotonic function of spontaneous tension and undergoes a sharp transition from large to small values as the magnitude of interfacial tension increases. We also predict that the physical properties of the RBC cytoskeleton layer - particularly the resting length of the cytoskeleton - play key roles in specifying the degree of the membrane wrapping. We also found that the shear energy of cytoskeleton deformation diverges at the full wrapping state, suggesting the local disassembly of the cytoskeleton is required to complete the merozoite entry. Additionally, using our theoretical framework, we predict the landscape of myosin-mediated forces and the physical properties of the RBC membrane in regulating successful malaria invasion. Our findings on the crucial role of RBC membrane tension in inhibiting malaria invasion can have implications for developing novel antimalarial therapeutic or vaccine-based strategies. SignificanceRBC membrane tension plays an important role in regulating RBC shape and functionality. In particular, recent experimental studies have shown that elevated RBC membrane tension protects against severe malaria infection. In this study, we sought to identify how different contributions to the the effective membrane tension can contribute to this mechanically-driven protection against malaria invasion. Using a mathematical model, we derived a relationship between the effective tension of the RBC membrane - comprising a lipid bilayer and a cytoskeleton layer- and the degree of membrane wrapping during malaria invasion. Our model shows that the shear resistance of the RBC cytoskeleton plays an important role in inhibiting malaria invasion. Our findings can be generalized to the role of cell membrane mechanics in many wrapping phenomena providing insight into the crucial contributions of the host-cell membrane in protection against severe infections.

biophysics↗

bioRxiv2023

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets.

biophysics↗