Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

The Interplay of Structural and Cellular Biophysics Controls Clustering of Multivalent Molecules

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. It is not well understood, however, how molecular structure and cellular organization control clustering. Using coarse-grained kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1, and the actin nucleation promoting factor NWASP. Steady-state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching) but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.     

Molecular Adsorption on Cold Gas-Phase Hydrogen-Bonded Clusters of Chiral Molecules

Origins of Life and Evolution of Biospheres - Gas-phase molecular adsorption was investigated as a model for molecular cloud formation. Molecular adsorption on cold gas-phase hydrogen-bonded...     

Formation and Dynamics of Topic Clusters in Free Recall

A. A. Smirnov was one of the first to study the problem of clustering of data in the process of remembering and recall, (3) as well as the multiform factors influencing clustering (the volume and difficulty of the material, the problem posed, the concern and individual characteristics of the subject). He demonstrated that clustering is an important tool for successful memorizing, and that clustering by meaning is a process that is often not fully conscious. Also significant is the fact established by Smirnov that "recall is more frequently accompanied by topic clustering than is remembering." (3; 244)     

Role of Water Molecules in Structure and Energetics of Pseudomonas aeruginosa Lectin I Interacting with Disaccharides

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an α-galactose residue at their nonreducing end, such as the disaccharides αGal1–2βGalOMe, αGal1–3βGalOMe, and αGal1–4βGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL·αGal1–2βGalOMe complex, which was solved at 2.4 Å resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1–2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.     

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Ab Initio Calculations of the Structure and Properties of Large Atomic Clusters

Abstract

A discussion is given of the problems involved in computing the total energy, using local density functional methods, of a cluster of atoms with a real space basis set of Gaussian orbitals. Particular attention is given to the methods used to evaluate the Hartree and exhange-correlation energies and their potentials. Several applications are described: molecular structures and properties, the bond lengths and dynamic properties of bulk silicon and diamond, the local vibratory mode of carbon in silicon, and the structures of H and H related complexes in diamond and gallium arsenide.     

Crowding Induces Complex Ergodic Diffusion and Dynamic Elongation of Large DNA Molecules

Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0–40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.     

Ensembles of Breathing Nucleosomes: A Computational Study

About three-fourths of the human DNA molecules are wrapped into nucleosomes, protein spools with DNA. Nucleosomes are highly dynamic, transiently exposing their DNA through spontaneous unspooling. Recent experiments allowed to observe the DNA of an ensemble of such breathing nucleosomes through x-ray diffraction with contrast matching between the solvent and the protein core. In this study, we calculate such an ensemble through a Monte Carlo simulation of a coarse-grained nucleosome model with sequence-dependent DNA mechanics. Our analysis gives detailed insights into the sequence dependence of nucleosome breathing observed in the experiment and allows us to determine the adsorption energy of the DNA bound to the protein core as a function of the ionic strength. Moreover, we predict the breathing behavior of other potentially interesting sequences and compare the findings to earlier related experiments.     

免疫突触的形成及其介导的分子识别

T细胞和APC细胞相互作用形成免疫突触涉及到连续发生的一系列的分子识别事件,最初APC细胞在趋化因子的作用下向T细胞移动,相遇后在抗原非依赖性的弱的黏附力作用下发生最初的黏附,同时伴随着TCR在APC表面俘获特异性抗原;抗原识别之后,由多种机制使T细胞和APC紧密接触并维持一段时间,随后分开,最终引起T细胞的增殖和分化。对免疫突触形成过程中的分子识别机制目前尚无定论,拓扑模式和数学模式的解释,脂筏和细胞骨架蛋白的重排以及接头蛋白的连接为免疫突触形成中分子的识别提供了一定的依据。     

Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids

Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.     

Regulated Formation of Extrachromosomal Circular DNA Molecules during Development in Xenopus laevis

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.     

Diffusion of Large Molecules into Assembling Nuclei Revealed Using an Optical Highlighting Technique

The nuclear envelope (NE) defines the nuclear compartment, and nuclear pore complexes (NPCs) on the NE form aqueous passages through which small water-soluble molecules can passively diffuse. It is well known that proteins smaller than 50 kDa can diffuse though NPCs, whereas proteins larger than 60 kDa rarely enter by passive diffusion. Little, however, is known about how this size cutoff develops as the NE reassembles and the nucleus expands. In 1987, a well-known study identified an efficient mechanism by which large diffusing proteins (>60 kDa) were excluded from the reassembling nucleus after mitosis. Since then, it has been generally accepted that after mitosis, newly formed nuclei completely exclude all proteins except those that are initially bound to the mitotic chromosomes and those that are selectively imported through NPCs. Here, the tetrameric complex of the photoconvertible fluorescent protein KikGR (∼103 kDa) was optically highlighted in the cytoplasm and followed to examine its entry into nuclei. Remarkably, highlighted complexes efficiently entered newly assembled nuclei during an ∼20-min period after the completion of cytokinesis. Because KikGR contains no known nuclear-localization or chromosome-binding sequences, our results indicate the diffusion barrier is less restrictive during nuclear reassembly.     

Interacting Factors Driving a Major Loss of Large Trees with Cavities in a Forest Ecosystem

Large trees with cavities provide critical ecological functions in forests worldwide, including vital nesting and denning resources for many species. However, many ecosystems are experiencing increasingly rapid loss of large trees or a failure to recruit new large trees or both. We quantify this problem in a globally iconic ecosystem in southeastern Australia – forests dominated by the world''s tallest angiosperms, Mountain Ash (Eucalyptus regnans). Tree, stand and landscape-level factors influencing the death and collapse of large living cavity trees and the decay and collapse of dead trees with cavities are documented using a suite of long-term datasets gathered between 1983 and 2011. The historical rate of tree mortality on unburned sites between 1997 and 2011 was >14% with a mortality spike in the driest period (2006–2009). Following a major wildfire in 2009, 79% of large living trees with cavities died and 57–100% of large dead trees were destroyed on burned sites. Repeated measurements between 1997 and 2011 revealed no recruitment of any new large trees with cavities on any of our unburned or burned sites. Transition probability matrices of large trees with cavities through increasingly decayed condition states projects a severe shortage of large trees with cavities by 2039 that will continue until at least 2067. This large cavity tree crisis in Mountain Ash forests is a product of: (1) the prolonged time required (>120 years) for initiation of cavities; and (2) repeated past wildfires and widespread logging operations. These latter factors have resulted in all landscapes being dominated by stands ≤72 years and just 1.16% of forest being unburned and unlogged. We discuss how the features that make Mountain Ash forests vulnerable to a decline in large tree abundance are shared with many forest types worldwide.