Thermal dependence of apolipoprotein A-I-phospholipid recombination

Studies of the recombination of apolipoprotein A-I (apo A-I), the major protein constituent of human high-density lipoprotein, and various synthetic phospholipids, both alone and in mixtures, have been performed. Pure diacyl phospholipids containing homologous fatty acids of the C12, C13, C14, and C15 series, as well as the two positional isomers containing C14 and C16 fatty acids in positions 1 and 2, undergo reaction with the apo A-I protein only near their gel-liquid-crystalline transition temperatures; the degree of reactivity of these phospholipids toward recombination was observed to decrease as the transition temperature increased. The presence of lysolecithin in the incubation mixtures at proportions of 5 mol/mol of protein or lower was not found to have a significant effect on the rate of recombination. Binary mixtures of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine at various proportions react maximally with apo A-I at the onset of the phase transition, as judged by comparison with published phase diagrams; in this case also, the rate of recombination was observed to decline for mixtures with higher phase transition temperatures. These results are interpreted in terms of protein insertion at lattice defects arising from the presence of phospholipid clusters undergoing the phase transition; these clusters are derived from the cooperative and simultaneous melting of a number of molecules, the cooperativity being dependent upon the nature of the phospholipid. It is postulated that phospholipids which melt in a more highly cooperative fashion are more capable of interacting with the apolipoproteins since these phospholipids will possess larger lattice defects during the phase transition.     

Conformational pathway for the kissing complex-->extended dimer transition of the SL1 stem-loop from genomic HIV-1 RNA as monitored by targeted molecular dynamics techniques

HIV-1 retroviral genomic RNA dimerization is initiated by loop-loop interactions between the SL1 stem-loops of two identical RNA molecules. The SL1-SL1 unstable resulting kissing complex (KC) then refolds irreversibly into a more stable complex called extended dimer (ED). Although the structures of both types of complex have been determined, very little is known about the conformational pathway corresponding to the transition, owing to the difficulty of observing experimentally intermediate conformations. In this study, we applied targeted molecular dynamics simulation techniques (TMD) to the phosphorus atoms for monitoring this pathway for the backbone, and a two-step strategy was adopted. In a first step, called TMD(-1), the dimer structure was constrained to progressively move away from KC without indicating the direction, until the RMSD from KC reaches 36A. A total of 20 TMD(-1) simulations were performed under different initial conditions and different simulation parameters. For RMSD ranging between 0 and 22A, the whole set of TMD(-1) simulations follows a similar pathway, then divergences are observed. None of the simulations leads to the ED structure. At RMSD=22A, the dimers look like two parallel Us, still linked by the initial loop-loop interaction, but the strands of the stems (the arms of the Us) are positioned in such a manner that they can form intramolecular as well as intermolecular Watson-Crick base-pairs. This family of structure is called UU. In a second step (TMD simulations), 18 structures were picked up along the pathways generated with TMD(-1) and were constrained to move toward ED by decreasing progressively their RMSD from ED. We found that only structures from the UU family are able to easily reach ED-like conformations of the backbones without exhibiting a large constraint energy.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.     

Nature of the stacking of nucleic acid bases in water: a Monte Carlo simulation

The results of a Monte Carlo simulation of the hydration of uracil and thymine molecules, their stacked dimers and hydrogen-bonded base pairs are presented. Simulations have been performed in a cluster approximation. The semiempirical atom-atom potential functions have been used (cluster consisting of 200 water molecules). It has been shown that the stacking interactions of uracil and thymine molecules in water arise mainly due to the increase in the water-water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than base pairs in water. This preference is mainly due to the energetically more favourable structure of water around the stack.     

Nature of the Stacking of Nucleic Acid Bases in Water: a Monte Carlo Simulation

Abstract

The results of a Monte Carlo simulation of the hydration of uracil and thymine molecules, their stacked dimers and hydrogen-bonded base pairs are presented. Simulations have been performed in a cluster approximation. The semiempirical atom-atom potential functions have been used (cluster consisting of 200 water molecules). It has been shown that the stacking interactions of uracil and thymine molecules in water arise mainly due to the increase in the water-water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than base pairs in water. This preference is mainly due to the energetically more favourable structure of water around the stack.     

Modeling the ion channel structure of cecropin.

Atomic-scale computer models were developed for how cecropin peptides may assemble in membranes to form two types of ion channels. The models are based on experimental data and physiochemical principles. Initially, cecropin peptides, in a helix-bend-helix motif, were arranged as antiparallel dimers to position conserved residues of adjacent monomers in contact. The dimers were postulated to bind to the membrane with the NH2-terminal helices sunken into the head-group layer and the COOH-terminal helices spanning the hydrophobic core. This causes a thinning of the top lipid layer of the membrane. A collection of the membrane bound dimers were then used to form the type I channel structure, with the pore formed by the transmembrane COOH-terminal helices. Type I channels were then assembled into a hexagonal lattice to explain the large number of peptides that bind to the bacterium. A concerted conformational change of a type I channel leads to the larger type II channel, in which the pore is formed by the NH2-terminal helices. By having the dimers move together, the NH2-terminal helices are inserted into the hydrophobic core without having to desolvate the charged residues. It is also shown how this could bring lipid head-groups into the pore lining.     

A novel hybrid simulation for study of multiscale phenomena

A novel multiscale model and simulation has been developed by combining the algorithms from a course grained 2D lattice model with Brownian dynamics (BD). The lattice model incorporates Lennard-Jones and coulombic interactions between particles, as well as nearest-neighbor interactions. The BD simulation allows the particles to move in solution before they adsorb to the surface. This hybrid simulation is used to study the behavior of nanoparticles in the presence of a surface and evaporation of solvent. The simulation is able to produce amorphous topologies of nanoparticles on the surface.     

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.     

Effects of oleic acid and its congeners,elaidic and stearic acids,on the structural properties of phosphatidylethanolamine membranes

Fatty acid derivatives are abundant in biological membranes, mainly as components of phospholipids and cholesterol esters. Their presence, free or bound to phospholipids, modulates the lipid membrane behavior. The present study shows the differential influence of the C-18 fatty acids (FAs), oleic, elaidic, and stearic acids on the structural properties of phosphatidylethanolamine (PE). X-ray diffraction of PE-FA systems demonstrated that oleic acid (OA) produced important concentration-dependent alterations of the lipid membrane structure: it induced reductions of up to 20-23 degrees C in the lamellar-to-hexagonal transition temperature of 1-palmitoyl-2-oleoyl PE and dielaidoyl PE and regulated the dimensions of the hexagonal lattice. In contrast, elaidic and stearic acids did not markedly alter the phospholipid mesomorphism. The above effects were attributed to the different "molecular shape" of OA (with a kink at the middle of the molecule) with respect to their congeners, elaidic and stearic acids. The effects of free fatty acids (FFAs) on membrane structure are relevant for several reasons: i) some biological membranes contain very high levels of FFAs. ii) Mediterranean diets with high OA intake have been shown to exert protective effects against tumoral and hypertensive pathologies. iii) FFA derivatives have been developed as antitumoral and antihypertensive drugs.     

The effect of the phase transition on the hydration and electrical conductivity of phospholipids.

Adsorption isotherms for various saturated phosphatidylcholines have been obtained. Lipids above and below their phase transition temperature differ only in the amount of water adsorbed and not in the nature of their adsorption isotherms. Cholesterol has an effect similar to that of increasing unsaturation in the hydrocarbon chains. Decreasing the length of the hydrocarbon chains for lipids below their phase transition temperature has no effect on the isotherms. If the chain length is short enough so that the lipids are above their transition temperature, however, a large increase in water adsorption occurs. All of the phospholipids exhibit a rapid increase of electrical conductivity for a few water molecules adsorbed per lipid molecule. All of the phospholipids show a saturation in conductivity at greater amounts of adsorbed water; the shape of the saturation region depends on whether the lipids are above or below their phase transition temperature. The activation energy for the electrical conductivity process depends on whether the hydrated lipids are in the "liquid-like" of the crystalline state, being lower for phospholipids in the liquid-like state. If the lipids are hydrated above their phase transition temperatures, their activation energies are lower than if they are hydrated below the transition temperature. Cholesterol lowers the activation energy. The phosphatidylcholines can be characterized by different activation energies, depending both upon their physical state and the presence of unsaturation in their hydrocarbon chains.     

Dimers of light-harvesting complex 2 from Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy

Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein complexes to be arranged in a densely packed energy-transducing network. Protein packing may play a determining role in the formation of functional photosynthetic domains and membrane curvature. To further investigate in detail the packing effects of like-protein photosynthetic complexes, we report an atomic force microscopy investigation on artificially created 2D crystals of the peripheral photosynthetic light-harvesting complexes 2 (LH2's) from the bacterium Rhodobacter sphaeroides. Instead of the usually observed one or two different crystallization lattices for one specific preparation protocol, we find seven different packing lattices. The most abundant crystal types all show a tilting of LH2. Most surprisingly, although LH2 is a monomeric protein complex in vivo, we find an LH2 dimer packing motif. We further characterize two different dimer configurations: in type 1, the LH2's are tilted inwards, and in type 2, they are tilted outwards. Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2's that constitute a lattice of zig-zagging LH2's. In addition, analyses of the tilt of the LH2's within the zig-zag lattice and that observed within the dimers corroborate their similar packing motifs. The type 2 dimer configuration exhibits a tilt that, in the absence of up-down packing, could bend the lipid bilayer, leading to the strong curvature of the LH2 domains as observed in Rhodobacter sphaeroides photosynthetic membranes in vivo.     

Coarse-grained lattice model simulations of sequence-structure fitness of a ribosome-inactivating protein

Many realistic protein-engineering design problems extend beyond the computational limits of what is considered practical when applying all-atom molecular-dynamics simulation methods. Lattice models provide computationally robust alternatives, yet most are regarded as too simplistic to accurately capture the details of complex designs. We revisit a coarse-grained lattice simulation model and demonstrate that a multiresolution modeling approach of reconstructing all-atom structures from lattice chains is of sufficient accuracy to resolve the comparability of sequence-structure modifications of the ricin A-chain (RTA) protein fold. For a modeled structure, the unfolding-folding transition temperature was calculated from the heat capacity using either the potential energy from the lattice model or the all-atom CHARMM19 force-field plus a generalized Born solvent approximation. We found, that despite the low-resolution modeling of conformational states, the potential energy functions were capable of detecting the relative change in the thermodynamic transition temperature that distinguishes between a protein design and the native RTA fold in excellent accord with reported experimental studies of thermal denaturation. A discussion is provided of different sequences fitted to the RTA fold and a possible unfolding model.     

Understanding a molecular motor walking along a microtubule: an asymmetric Brownian motor driven by bubble formation with a focus on binding affinity

In recent years, many studies on a molecular motor have been conducted in the fields of biorheology and nanoengineering. The molecular motor is a molecule that converts the chemical energy obtained by ATP hydrolysis into mechanical energy. Explaining this mechanism is important for nanoengineering. A kinesin, which is a type of molecular motor, has the characteristics to move on a microtubule with hand-over-hand steps. The kinesin walking behaviour is explained by the ‘asymmetric Brownian ratchet model’. Previously, we had suggested that the walking mechanism was achieved by the bubble formation in a nanosized channel surrounded by hydrophobic atoms with the transition between the two states – bubble state and liquid state. However, the walking behaviour of the model motor was different from that of a single molecule measurement of a kinesin. In this study, we constructed a new motor system focused on the asymmetric binding affinity of a motor protein and performed a model simulation using the dissipative particle dynamics method. As a result, it was observed that hand-over-hand walking depends on the transition position ratio and the transition frequency coefficient. Moreover, the efficiency of the new motor system is higher than that of the previous motor systems. The new motor model can provide a simulation guide for the design of biomimetic nanomachines.     

Straight GDP-tubulin protofilaments form in the presence of taxol

Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration.     

Beta-sheet coil transitions in a simple polypeptide model.

A simplified model of a polypeptide chain is used to study the dynamics of the beta-sheet-coil transition. Each amino acid residue is treated as a single quasiparticle in an effective potential that approximates the potential of mean force in solution. The model is used to study the equilibrium and dynamic aspects of the sheet-coil transition. Systems studied include ones with both strands free to move (two-strand sheet), and ones with either strand fixed in position (multistrand sheet). The equilibrium properties examined include sheet-coil equilibrium constants and their dependence on chain position. Dynamic properties are investigated by a stochastic simulation of the Brownian motion of the chain in its solvent surroundings. Time histories of the dihedral angles and residue-residue cross-strand distances are used to study the behavior of the sheet structure. Auto- and cross-correlation functions are calculated from the time histories with relaxation times of tens to hundreds of picoseconds. Sheet-coil rate constants of tens of ns-1 were found for the fixed strand cases.     

Phospholipids in small extracellular vesicles: emerging regulators of neurodegenerative diseases and cancer

Small extracellular vesicles (sEVs) are generated by almost all cell types. They have a bilayer membrane structure that is similar to cell membranes. Thus, the phospholipids contained in sEVs are the main components of cell membranes and function as structural support elements. However, as in-depth research on sEV membrane components is conducted, some phospholipids have been found to participate in cellular biological processes and function as targets for cell–cell communication. Currently, sEVs are being developed as part of drug delivery systems and diagnostic factors for various diseases, especially neurodegenerative diseases and cancer. An understanding of the physiological and pathological roles of sEV phospholipids in cellular processes is essential for their future medical application. In this review, the authors discuss phospholipid components in sEVs of different origins and summarize the roles of phospholipids in sEV biogenesis. The authors further collect the current knowledge on the functional roles of sEV phospholipids in cell–cell communication and bioactivities as signals regulating neurodegenerative diseases and cancer and the possibility of using sEV phospholipids as biomarkers or in drug delivery systems for cancer diagnosis and treatment. Knowledge of sEV phospholipids is important to help us identify directions for future studies.     

On the Characterization and Software Implementation of General Protein Lattice Models

Abstract models of proteins have been widely used as a practical means to computationally investigate general properties of the system. In lattice models any sterically feasible conformation is represented as a self-avoiding walk on a lattice, and residue types are limited in number. So far, only two- or three-dimensional lattices have been used. The inspection of the neighborhood of alpha carbons in the core of real proteins reveals that also lattices with higher coordination numbers, possibly in higher dimensional spaces, can be adopted. In this paper, a new general parametric lattice model for simplified protein conformations is proposed and investigated. It is shown how the supporting software can be consistently designed to let algorithms that operate on protein structures be implemented in a lattice-agnostic way. The necessary theoretical foundations are developed and organically presented, pinpointing the role of the concept of main directions in lattice-agnostic model handling. Subsequently, the model features across dimensions and lattice types are explored in tests performed on benchmark protein sequences, using a Python implementation. Simulations give insights on the use of square and triangular lattices in a range of dimensions. The trend of potential minimum for sequences of different lengths, varying the lattice dimension, is uncovered. Moreover, an extensive quantitative characterization of the usage of the so-called “move types” is reported for the first time. The proposed general framework for the development of lattice models is simple yet complete, and an object-oriented architecture can be proficiently employed for the supporting software, by designing ad-hoc classes. The proposed framework represents a new general viewpoint that potentially subsumes a number of solutions previously studied. The adoption of the described model pushes to look at protein structure issues from a more general and essential perspective, making computational investigations over simplified models more straightforward as well.     

Noninteracting local-structure model of folding and unfolding transition in globular proteins. II. Application to two-dimensional lattice proteins

The noninteracting local-structure model of the folding and unfolding transition in globular proteins, the formulation of which was given in the preceding paper, is applied to the analysis of the two-dimensional lattice model of proteins. The lattice model of proteins is a theoretical tool designed to study the statistical-mechanical aspect of the folding and unfolding transition. Its dynamics have been studied by a method of Monte Carlo simulation. The noninteracting local-structure model reproduces the equilibrium properties of the lattice model obtained previously by computer simulation remarkably well, when the specificity of the long-range interactions is strong. This observation indicates that the basic assumption of the noninteracting local-structure model is equivalent to the assumption of strong specificity of intramolecular interactions. It is argued that by assuming this strong specificity, we can emphasize the correct main paths of folding and unfolding transition. The way local structures grow and/or merge along the most probable path of folding in the lattice model is discussed by the noninteracting local-structure model.     

Reaction pathway for the quaternary structure change in hemoglobin

We perform a computer simulation of the quaternary structure change during the allosteric transition of hemoglobin. The simulation is based on a docking procedure by which αβ dimers of human hemoglobin are associated into tetramers after being rotated in various orientations. The stability of tetramers thus reconstituted is estimated from the values of a simplified energy function describing nonbonded interactions and from the area of the surface buried in dimer–dimer contacts (their interface area), which we take to represent stabilizing interactions and solvent contribution. A systematic analysis of tetramers reconstituted with twofold symmetry reveals that when the dimers have the R tertiary structure, only tetramers having R-like quaternary structures are stable. When the dimers have the T tertiary structure, they may associate into T-like tetramers or a variety of quaternary structures ranging from T to near R, thus tracing a plausible reaction pathway for the allosteric transition. We subject intermediates of this pathway to energy refinement with rigid αβ dimers. The refinement demonstrates that symmetrical structures are more stable than non symmetrical ones. A detailed analysis of dimer–dimer contacts in intermediates shows how close packing is maintained over large interfaces throughout the quaternary structure change, especially in the “switch region” of contact between the C helix of α-chains and the FG corner of β-chains.     

Kinesin walks the line: single motors observed by atomic force microscopy

Motor proteins of the kinesin family move actively along microtubules to transport cargo within cells. How exactly a single motor proceeds on the 13 narrow lanes or protofilaments of a microtubule has not been visualized directly, and there persists controversy on the relative position of the two kinesin heads in different nucleotide states. We have succeeded in imaging Kinesin-1 dimers immobilized on microtubules with single-head resolution by atomic force microscopy. Moreover, we could catch glimpses of single Kinesin-1 dimers in their motion along microtubules with nanometer resolution. We find in our experiments that frequently both heads of one dimer are microtubule-bound at submicromolar ATP concentrations. Furthermore, we could unambiguously resolve that both heads bind to the same protofilament, instead of straddling two, and remain on this track during processive movement.