Understanding endophilin-mediated endocytosis via coarse-grained molecular dynamics simulations

Biophysical Journal

Phosphate, an essential metabolite involved in numerous cellular functions, is taken up by proton-coupled phosphate transporters of plants and fungi within the major facilitator family. Similar phosphate transporters have been identified across a diverse range of biological entities, including various protozoan parasites linked to human diseases, breast cancer cells with increased phosphate requirements, and osteoclast-like cells engaged in bone resorption. Prior studies have proposed an overview of the functional cycle of a proton-driven phosphate transporter (PiPT), yet a comprehensive understanding of the proposed reaction pathways necessitates a closer examination of each elementary reaction step within an overall kinetic framework. In this work, we leverage kinetic network modeling in conjunction with a "bottom-up" molecular dynamics approach to show how such an approach can characterize the proton …

Journal of the American Chemical Society

Amino acid ionic liquids (AAILs) are promising green materials for CO2 capture and conversion due to their large chemical structural tunability. However, the structural understanding of the AAILs underlying the CO2 reaction dynamics remains uncertain. Herein, we examine the steric effects of AAIL anions with various chemical structures on CO2 capture behavior. Based on ab initio free-energy sampling, we assess reaction mechanisms for carbamate formation via a two-step reaction pathway with a zwitterion intermediate undergoing dynamic proton transfer. Our results show that free-energy barriers for carbamate formation can be significantly reduced as the degree of steric hindrance of the anions decreases. Further analyses reveal that reduced steric hindrance of anions causes markedly stronger intermolecular interactions between zwitterion and anions, leading to an increased kinetically favorable …

Biophysical Journal

The enormous impact on public health and the economy of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection encourages unprecedented research worldwide to eradicate the virus. The SARS-CoV-2 virion is composed of four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). The M protein is the most abundant structural protein in the virion surface. It plays a central role in assembly and budding processes, whereas the N protein packages the viral genomic RNA into a ribonucleoprotein complex and interacts with M at the assembly sites. Our study investigates the protein-protein and protein-lipid interactions of M and N proteins in biologically relevant complex membrane models. We performed atomistic molecular dynamics simulations using recent experimental structures of M and N proteins. We identified important …

bioRxiv

The HIV-1 assembly process begins with a newly synthesized Gag polyprotein being targeted to the inner leaflet of the plasma membrane of the infected cells to form immature viral particles. Gag–membrane interactions are mediated through the myristoylated (Myr) N-terminal matrix (MA) domain of Gag, which eventually multimerize on the membrane to form trimers and higher order oligomers. The study of the structure and dynamics of peripheral membrane proteins like MA has been challenging for both experimental and computational studies due to the complex transient dynamics of protein–membrane interactions. Although the roles of anionic phospholipids (PIP2, PS) and the Myr group in the membrane targeting and stable membrane binding of MA are now well-established, the cooperative interactions between the MA monomers and MA-membrane remain elusive in the context of viral assembly and release …

Cytoskeleton

Vasodilator‐stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly‐Pro region, a G‐actin‐binding domain (GAB), and an F‐actin‐binding domain (FAB). Together, the poly‐Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin–G‐actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly‐Pro region and GAB domain of VASP with profilin–actin, has been solved over a decade ago; however, a detailed structure of the FAB‐F‐actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that …

Biophysical Journal

During the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-genomic RNA (gRNA) interactions play a crucial role in the multimerization process, which is yet to be fully understood. We performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model applied to the simulated trajectories, is observed to be altered by implicit gRNA binding, as well as the multimerization state of the Gag. The lateral dynamics of our simulated membrane-bound Gag proteins, with and without gRNA binding, agree with prior experimental data and help to validate our simulation models and methods. The gRNA binding is observed to affect mainly the SP1 …

Biophysical Journal

Caveolin is a monotopic membrane protein, widely expressed in metazoa and responsible for enigmatic curved membrane structures known as caveolae. This functionality and capacity for membrane deformation are mediated by the assembly of caveolins into oligomeric structures. Recently, the first high-resolution structure of purified human caveolin assemblies, CAV8S, revealed a unique organization of 11 caveolin protomers arranged in a tightly packed, radially symmetric spiral disc of∼ 15 nm diameter and 3 nm thickness. Among several unusual features, one face and all sides of this disc are almost exclusively hydrophobic, suggesting that it inserts deeply into lipid bilayers, displacing∼ 250 phospholipids from the cytosolic leaflet. Here, using a combination of coarse-grained and atomistic simulations, we evaluate the biophysical characteristics and stability of this arrangement. Our observations strongly …

arXiv preprint arXiv:2404.07156

Applying an excess entropy scaling formalism to the coarse-grained (CG) dynamics of liquids, we discovered that missing rotational motions during the CG process are responsible for artificially accelerated CG dynamics. In the context of the dynamic representability between the fine-grained (FG) and CG dynamics, this work introduces the well-known Stokes-Einstein and Stokes-Einstein-Debye relations to unravel the rotational dynamics underlying FG trajectories, thereby allowing for an indirect evaluation of the effective rotations based only on the translational information at the reduced CG resolution. Since the representability issue in CG modeling limits a direct evaluation of the shear stress appearing in the Stokes-Einstein and Stokes-Einstein-Debye relations, we introduce a translational relaxation time as a proxy to employ these relations, and we demonstrate that these relations hold for the ambient conditions studied in our series of work. Additional theoretical links to our previous work are also established. First, we demonstrate that the effective hard sphere radius determined by the classical perturbation theory can approximate the complex hydrodynamic radius value reasonably well. Also, we present a simple derivation of an excess entropy scaling relationship for viscosity by estimating the elliptical integral of molecules. In turn, since the translational and rotational motions at the FG level are correlated to each other, we conclude that the "entropy-free" CG diffusion only depends on the shape of the reference molecule. Our results and analyses impart an alternative way of recovering the FG diffusion from the CG description by coupling the …

Biophysical Journal

Wednesday, February 14, 2024 537a lamellipodia. Actin filaments elongate asymmetrically, with the barbed (plus) end growing faster than the pointed (minus) end. Cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations attempt to provide a molecular basis for the difference in polymerization rates. Near-atomic resolution cryo-EM reconstructions of the barbed and pointed ends (Carman et al. 2023 Science 380, 1287–1292) attribute the difference in polymerization rates to the ‘‘flat’’conformation barbed end subunits, similar to interior subunits, and a ‘‘twisted’’conformation of pointed end subunits, like actin monomers. On the other hand, all-atom molecular dynamics (MD) simulations of actin filament ends (Zsolnay et al. 2020 PNAS 117: 30458–30464) found that the terminal subunits at both ends adopt a more ‘‘twisted’’conformation than the interior filaments. At the barbed end subunits interior …

Biophysical Journal

Sunday, February 11, 2024 11a either needing more dynamics information or losing structure details. In this work, we introduce a systematic and more generalized approach called K-means clustering coarse-graining (KMC-CG) to generate optimal CG mappings for highly coarse-grained biomolecules. This method builds on the earlier approach of essential dynamics coarse-graining (ED-CG). A key aspect of the new approach is the integration of both the structural and dynamic contributions of the biomolecule into one single residual. KMC-CG removes the sequence-dependent constraints of ED-CG, allowing it to explore a more extensive solution space and thus enabling the discovery of more physically optimal CG mappings. Significantly, the implementation of the K-means clustering algorithm can variationally optimize the CG mapping with efficiency and stability. This new method is evaluated in three cases …

Journal of the American Chemical Society

In acidic HZSM-5 zeolite, the reactivity of a methanol molecule interacting with the zeolite proton is amenable to modification via coadsorbing a stochiometric amount of an electron density donor E to form the [(E)(CH3OH)(HZ)] complex. The rate of the methanol in this complex undergoing dehydration to dimethyl ether was determined for a series of E with proton affinity (PA) ranging from 659 kJ mol–1 for C6F6 to 825 kJ mol–1 for C4H8O and was found to follow the expression: Ln(Rate) – Ln(RateN2) = β(PA – PAN2)γ, where E = N2 is the reference and β and γ are constants. This trend is probably due to the increased stability of the solvated proton in the [(E)(CH3OH)(HZ)] complex with increasing PA. Importantly, this is also observed in steady-state flow reactions when stoichiometric quantities of E are preadsorbed on the zeolite. As demonstrated with E being D2O, the effect on methanol reactivity diminishes when E …

Biophysical Journal

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid …

Biophysical Journal

Antibiotic resistance in bacteria is a global health crisis. MipA, a 238-residue integral membrane protein (IMP), confers antibiotic resistance in gramnegative bacteria, including various disease-causing pathogens. As a member of the OmpV family, MipA regulates antibiotic resistance by altering antibiotic flow through bacterial membranes. Genetically removing MipA leads to an enhancement in bacterial resistance to multiple antibiotics. However, despite its crucial role, the structure, dynamics, and drug transporter mechanism of MipA remain unclear. Our study utilizes deep learning techniques and Anton molecular dynamics (MD) to understand IMP structure, motions, and the drug transport mechanism. Combining AlphaFold2 (AF2)(Jumper et al. 2021, Nature 596, 583), AF2-alt (Del Alamo et al. 2021, Elife 11: 1) using shallow multiple sequence alignments to explore alternative conformational states of proteins, and …

Simulating chemically reactive phenomena such as proton transport on nanosecond to microsecond and beyond time- scales is a challenging task. Ab initio methods are unable to currently access these timescales routinely, and traditional molecular dynamics methods feature fixed bonding arrangements that cannot account for changes in the system’s bonding topology. The Mul- tiscale Reactive Molecular Dynamics (MS-RMD) method, as implemented in the Rapid Approach for Proton Transport and Other Reactions (RAPTOR) software package for the LAMMPS molecular dynamics code, offers a method to routinely sample longer timescale reactive simulation data with statistical precision. RAPTOR may also be interfaced with enhanced sampling methods to drive simulations towards the analysis of reactive rare events, and a number of collective variables (CVs) have been developed to facilitate this. Key advances to this methodology, including GPU acceleration efforts and novel CVs to model water wire formation are reviewed, along with recent applications of the method which demonstrate its versatility and robustness.

Proceedings of the National Academy of Sciences

Nuclear import and uncoating of the viral capsid are critical steps in the HIV-1 life cycle that serve to transport and release genomic material into the nucleus. Viral core import involves translocating the HIV-1 capsid at the nuclear pore complex (NPC). Notably, the central channel of the NPC appears to often accommodate and allow passage of intact HIV-1 capsid, though mechanistic details of the process remain to be fully understood. Here, we investigate the molecular interactions that operate in concert between the HIV-1 capsid and the NPC that regulate capsid translocation through the central channel. To this end, we develop a “bottom-up” coarse-grained (CG) model of the human NPC from recently released cryo-electron tomography structure and then construct composite membrane-embedded CG NPC models. We find that successful translocation from the cytoplasmic side to the NPC central channel is …

Biophysical Journal

FtsZ, a bacterial tubulin, plays a crucial role in cytokinesis process. Like tubulin, it also consists of two domains—N-terminal and C-terminal domains. FtsZ assembles to form single-stranded protofilaments that exhibit a dynamic phenomenon known as treadmilling—wherein FtsZ subunits add to one end of the filament, and some dissociate from the other end thereby resulting in an apparent unidirectional motion of the filament while single FtsZ units remain stationary. Although FtsZ polymerizes to form protofilaments, it was experimentally determined that the protein polymerizes cooperatively which is unique for single-stranded filaments. Due to cooperative polymerization, a unique conformational switch accompanies the process. Therefore, FtsZ has two well-characterized conformations based on its polymerization propensity− 1. R state, preferred by the monomeric FtsZ and 2. T state, preferred by the polymeric FtsZ …

The Journal of Physical Chemistry B

The room temperature ionic liquid (RTIL) air–liquid interface plays an important role in many applications. Herein, we present molecular dynamics simulation results for the air–liquid interface of a common RTIL, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [C4mim][NTf2]. To elucidate the effects of electronic polarizability and scaled-charge ions on the properties of the RTIL air–liquid interface, we employ three different kinds of force fields: a nonpolarizable force field (FF) with united ion charges (FixQ), a nonpolarizable FF with scaled-charge by 0.8 (ScaleQ), and a polarizable FF (Drude). To identify whether the ions reside at the interface or not, the method of identification of the truly interfacial molecules is used. The structural and dynamical properties in the interfacial, subinterfacial, and central layers are evaluated. In general for bulk liquids, the FixQ model predicts too-ordered structures and too …

Journal of the American Chemical Society

Amino acid ionic liquids have received increasing attention as ideal candidates for the CO2 chemisorption process. However, the underlying molecular mechanisms, especially those involving proton transfer, remain unclear. In this work, we elucidate the atomistic-level reaction mechanisms responsible for carbamate formation during CO2 capture by amino acid ionic liquids through explicit ab initio molecular dynamics augmented by well-tempered metadynamics. The resulting ab initio free-energy sampling reveals a two-step reaction pathway in which a zwitterion, initially formed from the reaction between the anion of serine and CO2, undergoes a kinetically facile intermolecular proton transfer to the O atom of the COO– moiety in the nearby serine. Further analysis reveals that the significantly reduced free-energy barriers are attributed to enhanced intermolecular interaction between the zwitterion and serine, thus …

The Journal of Chemical Physics

This paper series aims to establish a complete correspondence between fine-grained (FG) and coarse-grained (CG) dynamics by way of excess entropy scaling (introduced in Paper I). While Paper II successfully captured translational motions in CG systems using a hard sphere mapping, the absence of rotational motions in single-site CG models introduces differences between FG and CG dynamics. In this third paper, our objective is to faithfully recover atomistic diffusion coefficients from CG dynamics by incorporating rotational dynamics. By extracting FG rotational diffusion, we unravel, for the first time reported to our knowledge, a universality in excess entropy scaling between the rotational and translational diffusion. Once the missing rotational dynamics are integrated into the CG translational dynamics, an effective translationrotation coupling becomes essential. We propose two different approaches for …

Biophysical Journal

1617-Pos Contribution of desmoplakin isoforms to desmosome architecture Krishna A. Patel, Collin M. Ainslie, Alexa L. Mattheyses. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA. Desmosomes are cell-cell junctions that maintain the integrity of tissues that experience significant mechanical stress, such as the skin and heart. Desmosomal plaque proteins are connected to the intermediate filaments by desmoplakin (DP), and genetic variants in DP are associated with several skin and heart pathologies, including erythrokeratodermia and arrhythmogenic cardiomyopathy. DP contains three domains: an N-terminal plakin head, a central rod, and an intermediate filament-binding C-terminal tail. Furthermore, DP has three splice isoforms (DPI, DPIa, and DPII) that differ only in the length of the rod domain and have distinct tissue-specific expression …

Biophysical Journal

The use of fast in silico prediction methods for protein-ligand binding free energies holds significant promise for the initial phases of drug development. Numerous traditional physics-based models (e.g., implicit solvent models), however, tend to either neglect or heavily approximate entropic contributions to binding due to their computational complexity. Consequently, such methods often yield imprecise assessments of binding strength. Machine learning models provide accurate predictions and can often outperform physics-based models. They, however, are often prone to overfitting, and the interpretation of their results can be difficult. Physics-guided machine learning models combine the consistency of physics-based models with the accuracy of modern data-driven algorithms. This work integrates physics-based model conformational entropies into a graph convolutional network. We introduce a new neural …

Biophysical Journal

Dipalmitoylphosphatidylcholine (DPPC) is a common model system for exploring the phase behavior of Lung Surfactant-the lipid-protein layer coating of the alveolar interface that facilitates breathing by reducing the line tension. The availability of enantiomers of DPPC in purified form allows us to investigate the chiral nature of model lipid monolayers through fluorescence microscopy and traditional Langmuir thermodynamic techniques. We focus on mixtures of DPPC with cholesterol, hexadecanol (HD), and palmitic acid (PA), which have been previously studied and shown to form equilibrium morphologies over experimental time scales REF: Valtierrez et al., Sci. Adv. v: 8: 14, 2022). In this poster, we will assess the use of complementary image processing and analysis routines to measure the curvature of morphologies within these monolayers. The introduction of cholesterol within the monolayers stabilizes …

Biophysical Journal

450a Wednesday, February 14, 2024 applicability of our platform by studying a range of cell-derived native vesicles, which includes bacterial outer membrane vesicles, mammalian plasma membrane-derived GPMVs, ER-derived microsomes, and other physiological membrane vesicles. In each of these cases, the application of our platform enables direct detection and ID of endogenous integral and peripheral MP-complexes, along with the bound ligands and lipids. Applying this platform, we demonstrate our ability to screen ligands against specific bacterial MP-complexes that are key targets for developing antibiotics, directly from their native membrane, at their endogenous level of expression. In a separate application, we show how specific post-translational modifications regulate molecular assemblies of neuronal SNARE complexes. Together, we present a broadly applicable platform to study the molecular …

Biophysical Journal

1Department of Cell Biology, Yale University, New Haven, CT, USA, 2Department of Pharmacology, Yale University, New Haven, CT, USA. The local membrane environment plays a paramount role in regulating the biology of membrane proteins (MP). Standard detergent-based MP-solubilization strategies disrupt the native bilayer-environment, leading to a fundamental gap in our understanding of how nanoscale molecular organization of MPs regulates their ability to elicit cellular responses. Addressing this, we have developed a proteome-wide quantitative guide for high-throughput, spatiallyresolved extraction of MPs into tunable native nanodiscs of 8-20 nm diameter. This enables downstream structural/functional studies of target MPs directly from native membranes. Combining existing MAPs with in-house chaincontrolled polymers, we developed a high-throughput, fluorescence-based assay for rapid screening …

Biophysical Journal

The organization of membrane proteins (MP) and lipids in the cell membrane is vital for all living organisms. To study this, we have developed a novel tunable in-vitro native mass spectrometry (nMS) platform that allows direct detection of the organization of MPs and bound lipids from membranes. This method employs customizable lipid bilayers to quantitatively reveal how specific membrane compositions and biophysical properties, such as curvature, tension, and fluidity, influence the lipid binding and functional arrangement of MPs. Here, we apply this platform to understand the lipid specificity of a trafficking MP, VAMP2 across the organellar membrane and how that tunes its biological function. Following its trafficking pathway, we study VAMP2 directly from liposomes mimicking different organelle membranes encountered during its trafficking pathways, such as ER, Golgi, synaptic vesicle (SV), and PM. By directly …

Biophysical Journal

Desmosomes are junctional protein assemblies found in epithelial and cardiac tissue that assist in maintaining tissue plasticity and integrity. They are composed of transmembrane cadherins desmocollin and desmoglein and intracellular plaque proteins plakoglobin (PG), plakophilin (PKP), and desmoplakin (DP). DP consists of a C-terminal tail domain that anchors the desmosome to intermediate filaments, central rod domain, and N-terminal head that binds PG and PKP. Mutations in desmosomal proteins result in weakened cell-cell adhesion in skin and heart diseases. Desmosomes exist in two states, calcium-dependent and hyper-adhesive, and can modulate between the two in dynamic processes such as wound healing. However, it is unclear how desmosome architecture is altered in hyper-adhesion and how the architecture impacts desmosome function. Hyper-adhesion can be conferred through different …

Biophysical Journal

Desmosomes are cell-cell junctions that maintain the integrity of tissues that experience significant mechanical stress, such as the skin and heart. Desmosomal plaque proteins are connected to the intermediate filaments by desmoplakin (DP), and genetic variants in DP are associated with several skin and heart pathologies, including erythrokeratodermia and arrhythmogenic cardiomyopathy. DP contains three domains: an N-terminal plakin head, a central rod, and an intermediate filament-binding C-terminal tail. Furthermore, DP has three splice isoforms (DPI, DPIa, and DPII) that differ only in the length of the rod domain and have distinct tissue-specific expression patterns. DPI is the primary cardiac isoform and is present in all desmosomes, whereas DPIa and DPII are expressed in stratified epithelia. Although DP’s binding partners and spliced isoforms are known, their spatial arrangement and functional …

Biophysical Journal

484a Wednesday, February 14, 2024 and efficient method for measuring surface-bound molecule flexibility. This method involves attaching a fluorophore to the molecule’s end and monitoring emitted light polarization as the molecule fluctuates. Using confocal polarization microscopy, we track the average changes in orientation of the fluorophore, which serves as a proxy for the flexibility of labeled molecules. Rigid molecules display minimal changes in fluorophore orientation, whereas flexible ones exhibit more significant changes in orientation. Using this molecular flexibility measurement technique, we first conducted in vitro experiments using DNA oligos attached to giant unilamellar vesicles. We find that increasing the length of the oligo increases its measured flexibility, consistent with computational models of a fluctuating polymer attached to a surface. Next, we investigated the role of surrounding molecules …

Biophysical Journal

Podosomes are punctate actin-rich cytoskeletal structures used by dendritic cells and macrophages to sense the mechanical properties of the surrounding extracellular environment, helping them patrol for pathogens. Podosomes are sensitive to substrate stiffness and probe the extra-cellular matrix by polymerizing actin from the cap against the plasma membrane, exerting force onto the substrate and detecting the opposing forces. Cycles of polymerization and collapse result in height oscillations of the podosome core. Interestingly, podosomes form clusters that exhibit collective oscillatory behavior, resulting in podosome height waves in which a local group of podosomes sequentially increase and then decrease their F-actin content. A model to explain the emergence of such collective behavior was missing until now, and no method had been able to quantify the spatiotemporal propagation of collective podosome …

Biophysical Journal

Many cell functions require a concerted effort from multiple membrane proteins, for example, for signaling, cell division, and endocytosis. One contribution to their successful self-organization stems from the membrane deformations that these proteins induce. While the pairwise interaction potential of two membrane-deforming spheres has recently been measured, membrane-deformation-induced interactions have been predicted to be nonadditive, and hence their collective behavior cannot be deduced from this measurement. We here employ a colloidal model system consisting of adhesive spheres and giant unilamellar vesicles to test these predictions by measuring the interaction potential of the simplest case of three membrane-deforming, spherical particles. We quantify their interactions and arrangements and, for the first time, experimentally confirm and quantify the nonadditive nature of membrane-deformation …

Biophysical Journal

Comparative methods in molecular evolution and structural biology rely heavily upon the site-wise analysis of DNA sequence and protein structure, both static forms of information. However, it is widely accepted that protein function results from nanoscale nonrandom machine-like motions induced by evolutionarily conserved molecular interactions. Comparisons of molecular dynamics (MD) simulations conducted between homologous sites representative of different functional or mutational states can potentially identify local effects on binding interaction and protein evolution. In addition, comparisons of different (i.e., nonhomologous) sites within MD simulations could be employed to identify functional shifts in local time-coordinated dynamics indicative of logic gating within proteins. However, comparative MD analysis is challenged by the large fraction of protein motion caused by random thermal noise in the …

Biophysical Journal

Mechanotransduction, the conversion of mechanical forces into biochemical signals, is essential for tissue development and physiological maintenance. Drosophila embryogenesis is ideal to study mechanotransduction given the significant strain cells experience to drive tissue-scale deformations. We aimed to understand the relationship between mechanical strain and Ca 2+ spiking in epithelial cells during Drosophila gastrulation. Previous research highlights the role of mechanosensitive ion channels (MSCs) in facilitating Ca 2+-dependent mechanotransduction during the processes of egg activation and dorsal closure. Intrigued by these reports, we examined the frequency and distribution of Ca 2+ spikes during the mechanically intensive process of gastrulation. We discover dynamic Ca 2+ spiking patterns during gastrulation that notably colocalize with high-strain areas. Fluorescence, time-lapse microscopy …

Biophysical Journal

The N-terminal domain of the Huntingtin protein (htt), called N17, plays a crucial role in the physiology of Huntingtonʼs disease. N17 forms an amphipathic helix when in contact with membranes or in aggregate form. Studies have indicated that N17 is responsible for the localization of htt to various organelles in the cell, including the endoplasmic reticulum, mitochondria and Golgi apparatus. It has also been implicated in increasing the rate of htt aggregation through its tendency to self interact. Computational and experimental techniques have delineated a detailed picture of the interactions of N17 with planar membranes. However, recent reports have suggested that N17 has an affinity for curved membranes. Given the uneven and curved shapes of organelles, it is important to examine the mechanistic preferences that drive the preferential partitioning of N17 to curved membranes. We used coarse-grained …

Biophysical Journal

Many forms of biological matter exhibit similar water-responsive changes in their size and mechanical properties. Recent experiments on bacterial spores led to the formulation of a theory of the mechanics of the spores based on the dominant role of hydration forces [1]. The theory was particularly successful in predicting unusual mechanical properties including a strong nonlinear elasticity and a jamming-driven transition in mechanical properties at short timescales, both of which were confirmed by atomic force microscopy experiments on spores. The simplicity of the underlying assumptions of the theory suggests that the theory could be applicable to other types of biological matter that exhibit water-responsive behavior. Here we present atomic force microscopy measurements on cellulose-based materials that reproduced the strong nonlinear elasticity discovered originally with spores. Quantitative analysis of the …

Biophysical Journal

Proteins are molecular machines and to understand how they work, we need to understand how they move. New pump-probe time-resolved X-ray diffraction methods open up ways to initiate and observe protein motions with atomistic detail in crystals on biologically relevant timescales. However, the practical limitations of these experiments demand parallel development of effective molecular dynamics approaches to accelerate progress and extract meaning. Here, we establish robust and accurate methods for simulating dynamics in protein crystals, a nontrivial process requiring careful attention to equilibration, environmental composition, and choice of force fields. With more than seven milliseconds of sampling of a single chain, we identify critical factors controlling agreement between simulation and experiments and show that simulated motions recapitulate ligand-induced conformational changes. This work …

Biophysical Journal

Electric-field stimulated X-ray crystallography (EF-X) involves applying an electric-field pulse to a protein crystal and using time-resolved X-ray diffraction to observe the response to the applied field. In this way, the dynamics of proteins can be resolved with high spatial resolution on a sub-microsecond timescale, making it highly suitable for comparison with molecular dynamics (MD) simulations. We emulate the EF-X experiment using all-atom MD simulations of a protein crystal with an applied electric field. As a model system, we simulate a 3× 3× 3 supercell of a PDZ domain and an electric field of 1 MV/cm. We compare the consistency of the induced motions in simulation and experiment. The EF-X experiment provides a rich data set and a novel approach to validating MD simulation.

Biophysical Journal

The blood-brain barrier is a highly complex physiological barrier that separates the blood from the central nervous system to maintain the latter's biological equilibrium. LDL receptor-related protein 1 (LRP1) is a receptor involved in BBB transcytosis and can be used by physiological or artificially induced processes. LRP1 is critical for the trafficking of misfolded proteins such as amyloid β, hyper-phosphorylated tau, and α-synuclein. Understanding its structure and function is essential to fully understanding neurological diseases like Alzheimer's, Parkinson's, Huntington's, and other related dementias. LRP1 is a modular membrane protein composed of 4544 amino acids, around 1200 of which are involved in three long and flexible structures that contain coordinated calcium ions and are decorated with small sugar chains called glycans. These three flexible components are believed to have an active role in ligand …

Biophysical Journal

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. During infection, P. gingivalis evades the host immune system via a number of virulence factors, such as the PG0352 sialidase. Several reports implicate sialidases in bacterial complement resistance but the underlying molecular mechanism has been elusive. Many complement factors and regulatory proteins are sialylated (modified with glycans containing terminal sialic acids), which is essential to their function. We provide functional and structural evidence that PG0352 sialidase can disarm the complement system via desialylation of complement proteins and regulators. Biochemical analyses reveal PG0352 can desialylate human serum and complement factors, thereby protecting bacteria from serum killing, mediated by membrane attack complement (MAC) complex formation. Structural studies reveal PG0352 is a dual domain sialidase …

Biophysical Journal

Seminal fluid in humans contain approximately 1-3 mM zinc, more than 100-fold higher than the amount circulating throughout the human body. Zinc is both essential and the most abundant trace metal in the body; however, there is no clear understanding of why zinc is enriched in human semen. We hypothesized that this abundant zinc may influence the physiology of sperm between mating and fertilization. Most of the zinc in seminal fluid is bound to proteins; thus, using the fluorescent zinc indicator FluoZin-1 to quantify the readily available zinc in seminal fluid, we found that pig semen includes 9.6±0.4 μM labile zinc (N= 8), and 5.0±0.7 μM for human (N= 3). To determine whether seminal zinc is imported by mammalian sperm, we used the intracellular zinc indicator FluoZin3-AM to observe changes before and after application of zinc. We found that mouse sperm quickly import extracellular zinc in a concentration …