Packing Defects Research Articles

Numerical study on the dynamic behavior of circular honeycomb structure with concentrated filling inclusions defects

Circular honeycombs are extensively used to absorb impact energy considering its good energy absorption properties. Rather than focusing on the existing perfect honeycomb, effects of concentrated filling inclusions on the in-plane dynamic behavior of circular honeycomb structure are studied in detail by using finite element simulations. The crushing model of honeycomb under in-plane impact is established which then is used to investigate the influences of packing ratio, defect distribution area and impact velocity on the deformation mode and the plateau stress. Investigation results show that the deformation model of imperfect honeycomb can still be classified into three types: Quasi-static mode, transition mode and dynamic mode; However, the value of packing ratio and defect location may seriously influence localized deformed band of transition mode; The plateau stress of the honeycomb relies on the defect location except for the packing ratio; The filling inclusions defect concentrated in sub-region 2 is more beneficial to improve the energy-absorption capacity; Especially under middle or low impact velocities, it displays higher sensitivity; The crushing plateau stress of honeycomb with α = 0.36 and defect concentrated in sub-region 2 is even improved about 56.9 % under impact velocity 100 m/s. These results can provide valuable suggestions in the study and design of other honeycomb structures.

PackMem: A Versatile Tool to Compute and Visualize Interfacial Packing Defects in Lipid Bilayers

The analysis of the structural organization of lipid bilayers is generally performed across the direction normal to the bilayer/water interface, whereas the surface properties of the bilayer at the interface with water are often neglected. Here, we present PackMem, a bioinformatic tool that performs a topographic analysis of the bilayer surface from various molecular dynamics simulations. PackMem unifies and rationalizes previous analyses based on a Cartesian grid. The grid allows identification of surface regions defined as lipid-packing defects where lipids are loosely packed, leading to cavities in which aliphatic carbons are exposed to the solvent, either deep inside or close to the membrane surface. Examples are provided to show that the abundance of lipid-packing defects varies according to the temperature and to the bilayer composition. Because lipid-packing defects control the adsorption of peripheral proteins with hydrophobic insertions, PackMem is instrumental for us to understand and quantify the adhesive properties of biological membranes as well as their response to mechanical perturbations such as membrane deformation.

Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn-membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm ). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34-55 °C) and preserved in its homologs β- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16-20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress

SummaryCell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

Defects and Chirality in the Nanoparticle-Directed Assembly of Spherocylindrical Shells of Virus Coat Proteins.

Virus coat proteins of small isometric plant viruses readily assemble into symmetric, icosahedral cages encapsulating noncognate cargo, provided the cargo meets a minimal set of chemical and physical requirements. While this capability has been intensely explored for certain virus-enabled nanotechnologies, additional applications require lower symmetry than that of an icosahedron. Here, we show that the coat proteins of an icosahedral virus can efficiently assemble around metal nanorods into spherocylindrical closed shells with hexagonally close-packed bodies and icosahedral caps. Comparison of chiral angles and packing defects observed by in situ atomic force microscopy with those obtained from molecular dynamics models offers insight into the mechanism of growth, and the influence of stresses associated with intrinsic curvature and assembly pathways.

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion.

Mitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of insitu and invitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein.

Scalable measurements of tow architecture variability in braided ceramic composite tubes

AbstractThe performance of braided ceramic matrix composites has been shown to depend on the spatial arrangement of tows; therefore, a new class of tools is required to measure irregularities in the composite architecture for components with intricate geometries. We report a scalable and robust reconstruction technique built upon stereoscopic digital image correlation that is able to efficiently measure the position of tows in arbitrarily shaped composites. This method was applied to triaxially braided ceramic matrix composite tubes intended for use as nuclear fuel cladding, which revealed both long‐range “systematic” tow packing defects associated with the manufacturing process and short‐range “intrinsic” defects due to the braid architecture. These findings suggested that the character of tow spacing variation in braided composite tubes was substantially more complex than in planar woven composites. These measurements are expected to lead to improved processing of braided composites and to facilitate the design of statistically representative virtual specimens for finite element modeling.

Lipid packing defects and membrane charge control RAB GTPase recruitment.

Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical‐shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C‐terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.

On the Origin of Microtubules’ High-Pressure Sensitivity

For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix.

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine. Molecular dynamics simulations reproduce the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 310-helical H-bonds. These effects facilitate local helix bending at the glycine site and change the collective dynamics of the helix.

Specific Coating of Cellular Lipid Droplets by a Giant and Repetitive Amphipathic Helix

Lipid droplets (LDs) are dynamic organelles that play an essential role in cellular lipid homeostasis; however, the mechanisms of selective protein targeting to LDs to mediate their function are poorly understood. Many LD proteins interact with LDs via amphipathic helices (AHs), which can mediate direct and reversible binding to lipid surfaces. We use a uniquely long and monotonous AH, found in the mammalian LD protein perilipin 4, to probe the physico-chemical properties of the LD surface. We show that this AH is completely unstructured in solution but can adopt a highly helical conformation, over a length of 660 amino acids, when in contact with lipids. By the use of fluorescently-tagged AH variants, we show that LDs are relatively permissive for AH binding, suggesting a surface with abundant lipid packing defects, in agreement with predicted behavior of a phospholipid monolayer on a neutral lipid core (Bacle et al., 2017, Biophys J, 112:1417). Whereas a slight increase in the hydrophobicity of perilipin 4-derived AHs makes them more promiscuous for binding to other cellular compartments, perturbing the distribution of charged residues in the polar face causes a striking loss of LD targeting. These results suggest that the physico-chemistry of the perilipin 4 AH is exquisitely tuned to ensure LD specificity. In vitro, we find that the purified wild-type AH binds poorly to bilayer membranes. In contrast, it can interact efficiently with neutral lipids and is capable of forming small uniformly-coated oil droplets. Accordingly, overexpression of this AH in cells overcomes a decrease in LD stability associated with phospholipid depletion. We propose that by substituting for the phospholipid monolayer, perilipin 4 may be important for stabilization of LDs when phospholipids are limiting, for example during periods of LD growth.

Mechanism and Determinants of Amphipathic Helix-Containing Protein Targeting to Lipid Droplets

Cytosolic lipid droplets (LDs) are the main storage organelles for metabolic energy in most cells. They are unusual organelles that are bounded by a phospholipid monolayer and specific surface proteins, including key enzymes of lipid and energy metabolism. Proteins targeting LDs from the cytoplasm often contain amphipathic helices, but how they bind to LDs is not well understood. Combining computer simulations with experimental studies invitro and in cells, we uncover a general mechanism for targeting of cytosolic proteins to LDs: large hydrophobic residues of amphipathic helices detect and bind to large, persistent membrane packing defects that are unique to the LD surface. Surprisingly, amphipathic helices with large hydrophobic residues from many different proteins are capable of binding to LDs. This suggests that LD protein composition is additionally determined by mechanisms that selectively prevent proteins from binding LDs, such as macromolecular crowding at the LD surface.

Erlang flow of hydrophilic pore formation and closure events in a lipid bilayer during phase transition resulting from diffusion in the radius space.

The Smoluchowski equation with an energy profile of a special type and an assumed hydrophobic ("half") pore source term is used to describe the process of hydrophilic pore formation in a lipid bilayer at the gel-liquid phase transition. The source term reflects the occurrence of molecule packing defects in a lipid bilayer at phase transition. The time sequences of the pore formation and closure events are treated as non-stationary, second-order Erlang flows whose characteristics depend on the equation solution. The computed distributions of the time intervals between hydrophilic pores, and pore lifetimes agree with the previously published experimental interpulse interval and pulse duration histograms for the current fluctuations through planar bilayer membranes of DPPC immersed in a LiCl aqueous solution containing polyethylene glycol. Thus, the statistical analysis of pore formation and closure times leads us to conclude that firstly, the increased permeability of a lipid bilayer during the gel-liquid phase transition is accounted for by the emergence of additional hydrophobic defects in the heterogeneous structure of the bilayer and secondly, that the non-exponential distributions of the lipid channel closed and open times observed in experiments are evidence that the process of hydrophilic pore formation is not a one-step process but involves at least two dependent events.

Toroidal Condensates by Semiflexible Polymer Chains: Insights into Nucleation, Growth and Packing Defects.

Deciphering the principles of DNA condensation is important to understand problems such as genome packing and DNA compaction for delivery in gene therapy. DNA molecules condense into toroids and spindles upon the addition of multivalent ions. Nucleation of a loop in the semiflexible DNA chain is critical for both the toroid and spindle formation. To understand the structural differences in the nucleated loop, which cause bifurcation in the condensation pathways leading to toroid or spindle formation, we performed molecular dynamics simulations using a coarse-grained bead-spring polymer model. We find that the formation of a toroid or a spindle is correlated with the orientation of the chain segments close to the loop closure in the nucleated loop. Simulations show that toroids grow in size when spindles in solution interact with a pre-existing toroid and merge into it by spooling around the circumference of the toroid, forming multimolecular toroidal condensates. The merging of spindles with toroids is facile, indicating that this should be the dominant pathway through which the toroids grow in size. The Steinhardt bond order parameter analysis of the toroid cross section shows that the chains pack in a hexagonal fashion. In agreement with the experiments there are regions in the toroid with good hexagonal packing and also with considerable disorder. The disorder in packing is due to the defects, which are propagated during the growth of toroids. In addition to the well-known crossover defect, we have identified three other forms of defects, which perturb hexagonal packing. The new defects identified in the simulations are amenable to experimental verification.

Atomic-Force Microscopy Analyses on Dislocation in Extinction Bands of Poly(dodecamethylene terephthalate) Spherulites Solely Packed of Single-Crystal-Like Lamellae

This study, using atomic-force and polarized-optical light (AFM and POM) microscopies on the extinction banded spherulites of poly(dodecamethylene terephthalate) (P12T) at high Tc = 110 °C with a film thickness kept at 1–3 µm, has verified that banded spherulites can be composed of stacks of entirely single-crystal-like lamellae free of any twisting, flipping, or bending, and no branching of lamellae. Defects in the crystal packing of extinction bands are present in both intra-band and inter-band regions. The intra-band defects originate from the miss-match in spiral-circling into circular bands while the inter-band defects are in the interfaces between successive bands where single crystals in the ridge are jammed to deformation, then suddenly precipitate prior to initiating another cycle of banding. The fish-scale lamellae, at the initiation of a cycle, are orderly packed as terrace-like single crystals; conversely, near or on the defected regions, they are highly jammed or squeezed and deformed to beyond recognition of their original single-crystal nature.

Lateral Organization of Host Heterogeneous Raft‐like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c‐Src

AbstractMembrane‐bound c‐Src non‐receptor tyrosine kinase, unlike other acyl‐modified lipid‐anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale‐clusters upon membrane binding of the Myr‐Src (2–9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr‐Src (2–9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

Lateral Organization of Host Heterogeneous Raft-like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c-Src.

Membrane-bound c-Src non-receptor tyrosine kinase, unlike other acyl-modified lipid-anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale-clusters upon membrane binding of the Myr-Src (2-9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr-Src (2-9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

How geometric frustration shapes twisted fibres, inside and out: competing morphologies of chiral filament assembly.

Chirality frustrates and shapes the assembly of flexible filaments in rope-like, twisted bundles and fibres by introducing gradients of both filament shape (i.e. curvature) and packing throughout the structure. Previous models of chiral filament bundle formation have shown that this frustration gives rise to several distinct morphological responses, including self-limiting bundle widths, anisotropic domain (tape-like) formation and topological defects in the lateral inter-filament order. In this paper, we employ a combination of continuum elasticity theory and discrete filament bundle simulations to explore how these distinct morphological responses compete in the broader phase diagram of chiral filament assembly. We show that the most generic model of bundle formation exhibits at least four classes of equilibrium structure-finite-width, twisted bundles with isotropic and anisotropic shapes, with and without topological defects, as well as bulk phases of untwisted, columnar assembly (i.e. 'frustration escape'). These competing equilibrium morphologies are selected by only a relatively small number of parameters describing filament assembly: bundle surface energy, preferred chiral twist and stiffness of chiral filament interactions, and mechanical stiffness of filaments and their lateral interactions. Discrete filament bundle simulations test and verify continuum theory predictions for dependence of bundle structure (shape, size and packing defects of two-dimensional cross section) on these key parameters.

How do packing defects modify the cooperative motions in supercooled liquids?

We use molecular dynamic simulations to investigate the relation between the presence of packing defects in a glass-former and the spontaneous cooperative motions called dynamic heterogeneity. For that purpose we use a simple diatomic glass-former and add a small number of larger or smaller diatomic probes. The diluted probes modify locally the packing, inducing structural defects in the liquid, while we find that the number of defects is small enough not to disturb the average structure. We find that a small packing modification around a few molecules can deeply influence the dynamics of the whole liquid, when supercooled. When we use small probe molecules, the dynamics accelerates and the dynamic heterogeneity decreases. In contrast, for large probes the dynamics slows down and the dynamic heterogeneity increases. The induced heterogeneities and transport coefficient modification increase when the temperature decreases and disappear around the onset temperature of the cage dynamics.

The Role of Packing Defects in the Stability and Function of the Intramembrane Protease GlpG

Although proteins are efficiently packed, structural analysis has revealed that packing defects (i.e., voids, tunnels and pockets) are prevalent inside proteins despite their unfavorable contribution to protein stability. It has been speculated that those packing defects may be necessary for ligand binding, transport or conformational changes required for function. Despite the potential importance, the role of packing defects in the stability and function of proteins is not clearly understood especially for membrane proteins. Here, using the rhomboid intramembrane protease GlpG as a model, we test the hypotheses that 1) improving the packing through cavity-filling substitution can be a general way to stabilize a membrane protein, and 2) if packing defects are critical for function, it would be possible to lock the protein conformation into either inactive or constitutively active state through engineering of the cavities. We first identified packing defects in several rhomboid proteases from different origins by homology modeling, and mapped conserved cavities onto the structure of GlpG. Then we designed cavity-filling substitutions, and measured the stability and activity of those variants. Among the 18 “cavity-filling” substitutions tested, three were significantly destabilizing (ΔΔGoU > 1 kcal/mol), three were moderately stabilizing (ΔΔGoU =0.6∼1.0 kcal/mol), and the rest retained the stability to the level similar to wild-type. Among the three stabilizing substitutions, one was fully active while the other two were inactive. Those two inactivating substitutions did not involve the active-site residues but targeted the sites that belonged to the previously identified more flexible regions in GlpG. This result suggests that the packing defects may be required for the movement of structural elements of proteins, and implies that protein packing may have evolved for function through the delicate balance between stability and flexibility.