Results in Journal Biophysical Journal: 68,828

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Published: 22 September 2021
Yongzheng Zhu, Xuan Bai,
Published: 22 September 2021
Abstract:
A mesoscopic model with molecular resolution is presented for dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC) monolayer simulations at the air–water interface using many-body dissipative particle dynamics (MDPD). The parameterization scheme is rigorously based on reproducing the physical properties of water and alkane and the interfacial property of the phospholipid monolayer by comparison with experimental results. Using much less computing cost, the present MDPD simulations yield a similar surface pressure–area isotherm as well as similar pressure-related morphologies as all-atomistic simulations and experiments. Moreover, the compressibility modulus, order parameter of lipid tails, and thickness of the phospholipid monolayer are quantitatively in line with the all-atomistic simulations and experiments. This model also captures the sensitive changes in the pressure–area isotherms of mixed DPPC/POPC monolayers with altered mixing ratios, indicating that the model is promising for applications with complex natural phospholipid monolayers. These results demonstrate a significant improvement of quantitative phospholipid monolayer simulations over previous coarse-grained models.
P.T. O’Neil, V. Vasquez-Montes, L. Swint-Kruse, M.R. Baldwin, A.S. Ladokhin
Published: 20 September 2021
Abstract:
Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. While the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single cysteine variants labeled with the environmentally-sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.
Phuong Thuy Bui,
Published: 20 September 2021
Abstract:
After translation, nascent proteins must escape the ribosomal exit tunnel to attain complete folding to their native states. This escape process also frees up the ribosome tunnel for a new translation job. In this study, we investigate the impacts of energetic interactions between the ribosomal exit tunnel and nascent proteins on the protein escape process by molecular dynamics simulations using partially coarse-grained models which incorporate hydrophobic and electrostatic interactions of the ribosome tunnel of H. marismortui with nascent proteins. We find that, in general, attractive interactions slow down the protein escape process whereas repulsive interactions speed it up. For the small globular proteins considered, the median escape time correlates with both the number of hydrophobic residues, $Nh$, and the net charge, Q, of a nascent protein. A correlation coefficient exceeding 0.96 is found for the relation between the median escape time and a combined quantity of $Nh+5.9Q$, suggesting that it is about 6 times more efficient to modulate the escape time by changing the total charge than the number of hydrophobic residues. The estimated median escape times are found in the sub-millisecond to millisecond range, indicating that the escape does not delay the ribosome recycling. For various types of the tunnel model, with and without hydrophobic and electrostatic interactions, the escape time distribution always follows a simple diffusion model, which describes the escape process as a downhill drift of a Brownian particle, suggesting that nascent proteins escape along barrier-less pathways at the ribosome tunnel.
Mahroof Kalathingal, Takashi Sumikama, ,
Published: 20 September 2021
Abstract:
Spontaneous unidirectional, or vectorial, insertion of transmembrane peptides is a fundamental biophysical process for toxin and viral actions. Polytheonamide B (pTB) is a potent cytotoxic peptide with a β6.3-helical structure. Previous experimental studies revealed that the pTB inserts into the membrane in a vectorial fashion and forms a channel with its single molecular length long enough to span the membrane. Also, molecular dynamics simulation studies demonstrated that the pTB is prefolded in the aqueous solution. These are unique features of pTB since most of the peptide toxins form channels through oligomerization of transmembrane helices. Here, we performed all-atom molecular dynamics simulations to examine the dynamic mechanism of the vectorial insertion of pTB, providing underlying elementary processes of the membrane insertion of a prefolded single transmembrane peptide. We find that the insertion of pTB proceeds with only the local lateral compression of the membrane in three successive phases: "landing," "penetration," and "equilibration" phases. The free energy calculations using the replica-exchange umbrella sampling simulations present an energy cost of 4.3 kcal/mol at the membrane surface for the membrane insertion of pTB from bulk water. The trajectories of membrane insertion revealed that the insertion process can occur in two possible pathways, namely "trapped" and "untrapped" insertions: in some cases, pTB is trapped in the upper leaflet during the penetration phase. Our simulations demonstrated the importance of membrane anchoring by the hydrophobic N-terminal blocking group in the landing phase, leading to subsequent vectorial insertion.
Zhe Gou, Hengdi Zhang, Mehdi Abbasi,
Published: 18 September 2021
Abstract:
ATP release by red blood cells (RBCs) under shear stress (SS) plays a pivotal role in endothelial biochemical signaling cascades. The aim of this study is to investigate through numerical simulation how does RBCs spatio-temporal organization depend on flow and geometrical conditions to generate ATP patterns. Numerical simulations were conducted in a straight channel by considering both plasma and explicit presence of RBCs, their shape deformation and cell-cell interaction, as well as ATP release by RBCs. Two ATP release pathways through cell membrane are taken into account, pannexin 1 channel (Px1), sensitive to SS, and cystic fibrosis transmembrane conductance regulator (CFTR) which responds to cell deformation. Several flow and hematocrit conditions are explored. The problem is solved by lattice Boltzmann method. Application of SS to the RBC suspension triggers a nontrivial spatial RBC organization and ATP patterns. ATP localizes preferentially in the vicinity of cell-free layer close to channel wall. Conditions for maximal ATP release per cell are identified, which depend on vessel size and hematocrit Ht. Increasing further Ht beyond optimum enhances the total ATP release but should degrade oxygen transport capacity, a compromise between an efficient ATP release and minimal blood dissipation. Moreover, ATP is boosted in capillaries suggesting a vasomotor activity coordination throughout the resistance network.
Rebecca F. Alford, Patrick J. Fleming, Karen G. Fleming,
Published: 16 September 2021
Abstract:
(Biophysical Journal 118, 2042–2055; April 21, 2020)
Tianhao Wang, Shouqin Lü, Yinjing Hao, Zinan Su, , Yuhong Cui
Published: 15 September 2021
Abstract:
Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitro hepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high, but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations are not only consistent with experimental measurements using in vitro liver sinusoidal chip, but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.
Katherine N. Liu,
Published: 15 September 2021
Abstract:
In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.
Sara Mingu,
Published: 15 September 2021
Suvrajit Maji, Joachim Frank
Published: 9 September 2021
Abstract:
This article bemoans the demise of truly modular open-source image processing systems, such as SPIDER, in recent years' development of tools for three-dimensional reconstruction in cryo-electron microscopy. Instead, today's users have to rely on the functionality of software systems that have little or no transparency. As a consequence, users of such packages no longer gain a conceptual understanding and intuitive grasp of the analytical routes leading from the stream of input data to the final density map. Possible remedies of this situation with free software are discussed.
Published: 9 September 2021
Istvan Szundi, Stephanie G. Pitch, Eefei Chen, David L. Farrens,
Published: 9 September 2021
Abstract:
Styrene-maleic acid (SMA) copolymers solubilize biological membranes to form lipid nanoparticles (SMALPs) that contain membrane proteins surrounded by native lipids, thus enabling the use of a variety of biophysical techniques for structural and functional studies. The question of whether SMALPs provide a truly natural environment or SMA solubilization affects the functional properties of membrane proteins, however, remains open. We address this question by comparing the photoactivation kinetics of rhodopsin, a G protein-coupled receptor (GPCR) in the disc membranes of rod cells, in native membrane and SMALPs prepared at different molar ratios between SMA(3:1) and rhodopsin. Time-resolved absorption spectroscopy, combined with complex kinetic analysis, reveals kinetic and mechanistic differences between the native membrane and SMA-stabilized environment. The results suggest a range of molar ratios for nanoparticles suitable for kinetic studies.
Z.M. Ingram, D.A. Schneider, A.L. Lucius
Published: 9 September 2021
Abstract:
RNA polymerases execute the first step in gene expression, transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multi-subunit RNA polymerases (Pol I, Pol II, and Pol III). RNA Polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell's most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Further, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here we show that Pol I exhibits considerable variability in both kmax and K1/2 values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster kmax values (∼200 s-1) compared to the remaining seven positions (∼60 s-1). Additionally, the average K1/2 for ATP incorporation was found to be approximately three-fold higher compared to GTP suggesting Pol I has a tighter affinity for GTP compared to ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.
Florian Carlier, Tinh-Suong Nguyen, ,
Published: 9 September 2021
Abstract:
Repeat-induced point mutation (RIP) is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. RIP detects duplications (irrespective of their origin, specific sequence, coding capacity and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely-positioned repeats can induce mutation of the adjoining non-repetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.
Guangzhe Liu, Zhaorong Liu, , ,
Published: 9 September 2021
Abstract:
Flagellated bacteria swim by rotating a bundle of helical flagella, and commonly explore the surrounding environment in a "run-and-tumble" motility mode. Here, we show that the upcoming flow could impact the bacterial run-and-tumble behavior by affecting the formation and dispersal of the flagellar bundle. Using a dual optical tweezers setup to trap individual bacteria, we characterized the effects of the imposed fluid flow and cell body rotation on the run-and-tumble behavior. We found that the two factors affect the behavior differently, with the imposed fluid flow increasing the running time and decreasing the tumbling time, while the cell body rotation decreasing the tumbling time only. Using numerical simulations, we computed the flagellar bundling time as a function of flow velocity, which agrees well with our experimental observations. The mechanical effects we characterized here provide novel ingredients for further studies of bacterial chemotaxis in complex environments such as dynamic fluid environments. SIGNIFICANCE A hallmark of bacterial chemotaxis is the "run-and-tumble" behavior. It remains unclear whether and how external mechanical factors affect this behavior. Here, we used dual optical tweezers to trap individual bacteria horizontally, and studied the run-and-tumble behavior under well-controlled mechanical conditions. We discovered that the mechanical factors such as imposed fluid flow and cell body rotation have a large impact on the run-and-tumble behavior through affecting flagellar bundle formation and dispersal. These mechanical effects provided novel insights for further studies of bacterial chemotaxis in complex environments.
, , Sang Joon Ahn, Alvin Yu, Alexander Groisman, Gregory A. Voth, Martin A. Schwartz
Published: 9 September 2021
Abstract:
Conversion of integrins from low to high affinity states, termed activation, is important in biological processes including immunity, hemostasis, angiogenesis and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. While it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here we address the role of integrin conformational activation in cellular mechanosensing. Comparison of WT vs activating mutants of integrin αVβ3 show that activating mutants shift cell spreading, FAK activation, traction stress and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the β3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild type integrin αVβ3 displaces under force, and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.
Aaron D. Rosenbloom, Elizabeth W. Kovar, David R. Kovar, Leslie M. Loew,
Published: 9 September 2021
Abstract:
We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments, but also considered other pathways. We aimed to identify the rate constants for these reactions that best fits experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work, rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.
Carine Julien,
Published: 9 September 2021
Abstract:
Evanescent-wave scattering is a topic in classical electrodynamics and in the study of colloidal particles near a boundary. However, how such near-surface scattering at sub-cellular refractive-index heterogeneities degrades the excitation confinement in biological total internal reflection fluorescence microscopy has not been well studied. An elegant theoretical work by Axelrod and Axelrod now addresses this very relevant question and reveals that - even when scattered - evanescent light preserves some of its surprising optical properties.
Mark E. Zweifel, Laura A. Sherer, Biswaprakash Mahanta,
Published: 7 September 2021
Abstract:
Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin's nucleation activity. Sustained elongation of small numbers of formin-bound filaments therefore requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin's nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.
, Žiga Pandur, Samo Penič, Aleš Iglič, Veronika Kralj-Iglič, David Stopar
Published: 7 September 2021
Abstract:
It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work we identify lipid vesicle shape dynamics prior to the solubilization of DOPC giant vesicles with Triton X-100 detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A 3D Monte-Carlo scheme was constructed that emulated the non-equilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of Triton X-100 detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the Triton X-100 molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules that is nonequilibrium in nature and very difficult to study.
Published: 7 September 2021
Abstract:
A nanoparticle (NP) immersed in biological media rapidly forms a corona of adsorbed proteins, which later controls the eventual fate of the particle and the route through which adverse outcomes may occur. The composition and timescale for the formation of this corona are both highly dependent on both the NP and its environment. The deposition of proteins on the surface of the NP can be imitated by a process processes of random sequential adsorption and, based on this model, we develop a rate-equation treatment for the formation of a corona represented by hard spheres on spherical and cylindrical NPs. We find that the geometry of the NP significantly alters the composition of the corona through a process independent of the rate constants assumed for adsorption and desorption of proteins, with the radius and shape of the NP both influencing the corona. We further investigate the roles of protein mobility on the surface of the NP and changes in the concentration of proteins.
Matthew D. McCoy, John Hamre, Dmitri K. Klimov,
Published: 3 September 2021
Abstract:
(Biophysical Journal 120, 189–204; January 19, 2021)
, Laurent Schwartz
Published: 3 September 2021
Abstract:
We propose a simple mechanism for the self-replication of protocells. Our main hypothesis is that the amphiphilic molecules composing the membrane bilayer are synthesized inside the protocell through exothermic chemical reactions. The slow increase of the inner temperature forces the hottest molecules to move from the inner leaflet to the outer leaflet of the bilayer. Because of this outward translocation flow, the outer leaflet grows faster than the inner leaflet. This differential growth increases the mean curvature and amplifies any local shrinking of the protocell until it splits in two. The proposed model, based on mere laws of physics, is a step in the study of the origin of life, as well as a clue for a better understanding of cell proliferation in cancer.
Atsushi Matsuda,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3628-3640; https://doi.org/10.1016/j.bpj.2021.07.025

Published: 1 September 2021
Abstract:
Recent studies proposed that enzyme active sites induce evolutionary constraints at long distances. The physical origin of such long-range evolutionary coupling is unknown. Here, I use a recent biophysical model of evolution to study the relationship between physical and evolutionary couplings on a diverse data set of monomeric enzymes. I show that evolutionary coupling is not universally long-range. Rather, range varies widely among enzymes, from 2Å to 20Å. Furthermore, the evolutionary coupling range of an enzyme does not inform on the underlying physical coupling, which is short-range for all enzymes. Rather, evolutionary coupling range is determined by functional selection pressure. SIGNIFICANCE Until recently, only residues near enzyme active sites were thought to be evolutionarily constrained. However, recent studies proposed that active sites induce long-range evolutionary constraints. This seems to conflict with the common finding that physical couplings in proteins are short-range. This raises the question of how short-range physical couplings may cause long-range evolutionary couplings. Here, I show that the function that maps physical coupling into evolutionary coupling depends on functional selection pressure. Under weak selection, both couplings are similarly short-range; under strong selection, short-range physical coupling is non-linearly turned into long-range evolutionary coupling. Thus, due to a huge variation of selection pressure, evolutionary coupling range varies widely among enzymes, from very short (2 Å) to very long (20 Å).
S. Zhu, E. Kachooei, J.R. Harmer, L.J. Brown, F. Separovic,
Published: 1 September 2021
Abstract:
The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures provide sufficient signal-to-noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC spin-labelled Mac1 analogues were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labelled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the EPR electron-electron distance for the doubly spin-labelled peptide was ca. 1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol/carbonyl backbone and was stronger for the doubly spin-labelled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations, while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.
, Heather E. Findlay, Nicholas J. Brooks, Sowmya Purushothaman, Oscar Ces,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3787-3794; https://doi.org/10.1016/j.bpj.2021.06.038

Abstract:
Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend; features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC:DOPE:DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.
Natalia Ostrowska, Michael Feig,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3795-3806; https://doi.org/10.1016/j.bpj.2021.07.008

Abstract:
Using molecular dynamics simulations, we describe how crowded environments affect the internal dynamics and diffusion of the hepatitis C virus proteases NS3/4A. This protease plays a key role in viral replication and is successfully used as a target for antiviral treatment. The NS3 enzyme requires a peptide cofactor, called NS4A, with its central part interacting with the NS3 β-sheet, and flexible, protruding terminal tails that are unstructured in water solution. The simulations describe the enzyme and water molecules at atomistic resolution, whereas crowders are modeled via either all-atom or coarse-grained models to emphasize different aspects of crowding. Crowders reflect the polyethylene glycol (PEG) molecules used in the experiments to mimic the crowded surrounding. A bead-shell model of folded coarse-grained PEG molecules considers mainly the excluded volume effect, whereas all-atom PEG models afford more protein-like crowder interactions. Circular dichroism spectroscopy experiments of the NS4A N-terminal tail show that a helical structure is formed in the presence of PEG crowders. The simulations suggest that crowding may assist in the formation of an NS4A helical fragment, positioned exactly where a transmembrane helix would fold upon the NS4A contact with the membrane. In addition, partially interactive PEGs help the NS4A N-tail to detach from the protease surface, thus enabling the process of helix insertion and potentially helping the virus establish a replication machinery needed to produce new viruses. Results point to an active role of crowding in assisting structural changes in disordered protein fragments that are necessary for their biological function.
Federico Sebastiani, Hanna Michlits, Bettina Lier, Maurizio Becucci, Paul G. Furtmüller, Chris Oostenbrink, Christian Obinger, Stefan Hofbauer, Giulietta Smulevich
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3600-3614; https://doi.org/10.1016/j.bpj.2021.06.042

, Semen Yesylevskyy,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3776-3786; https://doi.org/10.1016/j.bpj.2021.06.039

Abstract:
Identification, visualization, and quantitation of cardiolipin (CL) in biological membranes is of great interest because of the important structural and physiological roles of this lipid. Selective fluorescent detection of CL using noncovalently bound fluorophore 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenylethene (TTAPE-Me) has been recently proposed. However, this dye was only tested on wild-type mitochondria or liposomes containing negligible amounts of other anionic lipids, such as phosphatidylglycerol (PG) and phosphatidylserine (PS). No clear preference of TTAPE-Me for binding to CL compared to PG and PS was found in our experiments on artificial liposomes, Escherichia coli inside-out vesicles, or Saccharomyces cerevisiae mitochondria in vitro or in situ, respectively. The shapes of the emission spectra for these anionic phospholipids were also found to be indistinguishable. Thus, TTAPE-Me is not suitable for detection, visualization, and localization of CL in the presence of other anionic lipids present in substantial physiological amounts. Our experiments and complementary molecular dynamics simulations suggest that fluorescence intensity of TTAPE-Me is regulated by dynamic equilibrium between emitting dye aggregates, stabilized by unspecific but thermodynamically favorable electrostatic interactions with anionic lipids, and nonemitting dye monomers. These results should be taken into consideration when interpreting past and future results of CL detection and localization studies with this probe in vitro and in vivo. Provided methodology emphasizes minimal experimental requirements, which should be considered as a guideline during the development of novel lipid-specific probes.
Published: 1 September 2021
Aparna Vilas Dongre, Sudip Das, Asutosh Bellur, Sanjeev Kumar, Anusha Chandrashekarmath, Tarak Karmakar, Padmanabhan Balaram, , Hemalatha Balaram
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3732-3746; https://doi.org/10.1016/j.bpj.2021.07.014

Abstract:
Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.
Madhura De, Mehmet Ali Öztürk, Sebastian Isbaner, ,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3747-3763; https://doi.org/10.1016/j.bpj.2021.07.012

Abstract:
Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences.
Published: 1 September 2021
Published: 1 September 2021
Abstract:
Biophysical modelling of development started with Alan Turing. His two-morphogen reaction-diffusion (RD) model was a radical but powerful simplification. Despite its apparent limitations, the model captured real developmental processes that, only recently, have been validated at the molecular level in many systems. Precision and robustness of RD patterning, despite boundary condition-dependence, remain active areas of investigation in developmental biology.
Published: 1 September 2021
Abstract:
Intrinsically disordered proteins and protein regions make up a substantial fraction of many proteomes where they play a wide variety of essential roles. A critical first step in understanding the role of disordered protein regions in biological function is to identify those disordered regions correctly. Computational methods for disorder prediction have emerged as a core set of tools to guide experiments, interpret results, and develop hypotheses. Given the multiple different predictors available, consensus scores have emerged as a popular approach to mitigate biases or limitations of any single method. Consensus scores integrate the outcome of multiple independent disorder predictors and provide a per-residue value that reflects the number of tools that predict a residue to be disordered. Although consensus scores help mitigate the inherent problems of using any single disorder predictor, they are computationally expensive to generate. They also necessitate the installation of multiple different software tools, which can be prohibitively difficult. To address this challenge, we developed a deep-learning-based predictor of consensus disorder scores. Our predictor, metapredict, utilizes a bidirectional recurrent neural network trained on the consensus disorder scores from 12 proteomes. By benchmarking metapredict using two orthogonal approaches, we found that metapredict is among the most accurate disorder predictors currently available. Metapredict is also remarkably fast, enabling proteome-scale disorder prediction in minutes. Importantly, metapredict is fully open source and is distributed as a Python package, a collection of command-line tools, and a web server, maximizing the potential practical utility of the predictor. We believe metapredict offers a convenient, accessible, accurate, and high-performance predictor for single-proteins and proteomes alike.
Daniel Klose, Andrea Holla, Christoph Gmeiner, Daniel Nettels, Irina Ritsch, Nadja Bross, Maxim Yulikov, Frédéric H.-T Allain, Benjamin Schuler, Gunnar Jeschke
Published: 1 September 2021
Amer Mansour, Jonathan B. Sellon, Daniel Filizzola, Roozbeh Ghaffari, Mary Ann Cheatham, Dennis M. Freeman
Published: 1 September 2021
Jintong Lang, Qing Nie, Chunhe Li
Published: 1 September 2021
Anna Sinelnikova, Thomas Mandl, Harald Agelii, Oscar Grånäs, , ,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3709-3717; https://doi.org/10.1016/j.bpj.2021.07.017

Abstract:
Proteins often have nonzero electric dipole moments, making them interact with external electric fields and offering a means for controlling their orientation. One application that is known to benefit from orientation control is single-particle imaging with x-ray free-electron lasers, in which diffraction is recorded from proteins in the gas phase to determine their structures. To this point, theoretical investigations into this phenomenon have assumed that the field experienced by the proteins is constant or a perfect step function, whereas any real-world pulse will be smooth. Here, we explore the possibility of orienting gas-phase proteins using time-dependent electric fields. We performed ab initio simulations to estimate the field strength required to break protein bonds, with 45 V/nm as a breaking point value. We then simulated ubiquitin in time-dependent electric fields using classical molecular dynamics. The minimal field strength required for orientation within 10 ns was on the order of 0.5 V/nm. Although high fields can be destructive for the structure, the structures in our simulations were preserved until orientation was achieved regardless of field strength, a principle we denote "orientation before destruction."
Jon Pallbo, Ulf Olsson, Emma Sparr
Published: 1 September 2021
Yousra El Ghaleb, Monica L. Fernández-Quintero, Stefania Monteleone, Petronel Tuluc, Marta Campiglio, Klaus R. Liedl,
Published: 1 September 2021
Fusheng Liu, Hou Wu, Xiaoyu Yang, Yuqin Dong, Guoyou Huang, Guy M. Genin, ,
Published: 1 September 2021
Biophysical Journal, Volume 120, pp 3764-3775; https://doi.org/10.1016/j.bpj.2021.06.040

Abstract:
Although coupling between cardiomyocytes and myofibroblasts is well known to affect the physiology and pathophysiology of cardiac tissues across species, relating these observations to humans is challenging because the effect of this coupling varies across species and because the sources of these effects are not known. To identify the sources of cross-species variation, we built upon previous mathematical models of myofibroblast electrophysiology and developed a mechanoelectrical model of cardiomyocyte-myofibroblast interactions as mediated by electrotonic coupling and transforming growth factor-β1. The model, as verified by experimental data from the literature, predicted that both electrotonic coupling and transforming growth factor-β1 interaction between myocytes and myofibroblast prolonged action potential in rat myocytes but shortened action potential in human myocytes. This variance could be explained by differences in the transient outward K+ current associated with differential Kv4.2 gene expression across species. Results are useful for efforts to extrapolate the results of animal models to the predicted effects in humans and point to potential therapeutic targets for fibrotic cardiomyopathy.
Kerbaï Saïd Eroumé, , , , Aurélie Carlier
Published: 1 September 2021
Phansiri Boonnoy, Viwan Jarerattanachat, ,
Published: 31 August 2021
Abstract:
We performed a series of molecular dynamics (MD) simulations of cholesterol (Chol) in non-oxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC:Oxidized lipids bilayers with 0-50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased MD simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration lead to a change of phase state from the liquid-disordered (Ld) to the liquid-ordered (Lo) phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol's flip-flop rate depends on the lipid bilayer type and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.
Published: 31 August 2021
Abstract:
Molecular recognition is fundamental to transcription regulation. As a transcription factor, the tumor suppressor p53 has to recognize either specific DNA sequences or repressor protein partners. However, the molecular mechanism underlying the p53 conformational switch from the DNA-bound to repressor-bound states is not fully characterized. The highly charged nature of these interacting molecules prompted us to explore the non-bonded energy contributions behind molecular recognition of either a DNA or the repressor protein iASPP by p53 DNA-binding domain (p53DBD), using molecular dynamics (MD) simulation followed by rigorous analyses of energy terms. Our results illuminate the allosteric pathway by which iASPP binding to p53 diminishes binding affinity between p53 and DNA. Even though the p53DBD uses a common framework of residues for recognizing both DNA and iASPP, a comparison of the electrostatics in the two p53DBD complexes revealed significant differences in residue-wise contributions to the electrostatic energy. We found that an electrostatic allosteric communication path exists in the presence of both substrates. It consists of evolutionarily conserved residues, from residue K120 of the binding loop L1 to a distal residue R213 of p53DBD. K120 is near the DNA in the p53DBD-DNA complex, whereas iASPP binding moves it away from its DNA-binding position in the p53DBD-iASPP complex. The ‘energy-Hubs' (the residues show a higher degree of connectivity with other residues in the electrostatic networks) determined from the electrostatic network analysis established that this conformational change in K120 completely rewires the electrostatic network from K120 to R213, thereby impeding DNA binding. Furthermore, we found shifting populations of hydrogen bonds and salt bridges reduce pair-wise electrostatic energies within p53DBD in its DNA-bound state.
Jaya Krishna Koneru, Dube Dheeraj Prakashchand, Namita Dube, Pushpita Ghosh,
Published: 31 August 2021
Abstract:
Amphiphilic β-peptides, which are synthetically designed short-chain helical foldamer of β-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is: how does the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane disruption ability? Here, we address this question via a combination of all atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical β-peptide at wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide-to-lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pore via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid head groups, membrane thinning and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that, despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves towards the distal leaflet in favour of potential contact with polar head groups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same β-peptide, indicating strong sequence dependence. Taken together, this work put forward key perspectives of membrane-activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.
Koichiro Tashiro, Yasuhiro Shobayashi, Iku Ota,
Published: 31 August 2021
Abstract:
Mechanical thrombectomy has become the standard treatment for patients with an acute ischemic stroke. In this approach, to remove blood clots, mechanical force is applied using thrombectomy devices, where the interaction between the clot and the device could significantly affect the clot retrieval performance. It is expected that the finite element method (FEM) could visualize the mechanical interaction by the visualization of the stress transmission from the device to the clot. This research was aimed to verify the constitutive theory by implementing FEM based on the visco-hyperelastic theory, using a three-dimensional clot model. We used the visco-hyperelastic FEM to reproduce the mechanical behavior of blood clots, as observed in experiments. This study is focused on the mechanical responses of clots under tensile loading and unloading, because in mechanical thrombectomy, elongation is assumed to occur locally on the clots during the retrieval process. Several types of cylindrical clots were created by changing the fibrinogen dose. Tensile testing revealed that the stiffness (E0.45 value) of clots with fibrinogen could be more than three times higher than that of clots without fibrinogen. It was also found that the stiffness was not proportional to the fibrinogen dose. By fitting to the theoretical curve, it was revealed that the Mooney–Rivlin model could reproduce the hyperelastic characteristics of clots well. From the stress relaxation data, the three-chain Maxwell model could accurately fit the experimental viscoelastic data. FEM, taking the theoretical models into account, was then carried out, and the results matched well with the experimental visco-hyperelastic characteristics of clots under tensile load, reproducing the mechanical hysteresis during unloading, the stress dependence on the strain rate, and the time-dependent stress decrease in the stress relaxation test.
Eva-Maria E. Uhlemann, , ,
Published: 27 August 2021
Abstract:
ATP7A and ATP7B are structurally similar but functionally distinct active copper transporters that regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. Both proteins have a chain of six cytosolic metal-binding domains (MBDs) believed to be involved in the copper-dependent regulation of the activity and intracellular localization of these enzymes. While all the metal-binding domains are quite similar in structure, their spacing differs markedly between ATP7A and ATP7B. We show by NMR that the long polypeptide between MBD1 and MBD2 of ATP7A forms an additional, seventh, metastable domain HMA1A. The structure of HMA1A resembles the metal binding domains, but contains no copper binding site. The HMA1A domain, which is unique to ATP7A, may modulate regulatory interactions between MBD1-3, contributing to the distinct functional properties of ATP7A and ATP7B.
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