Macromolecular Solutions Research Articles

CRANK: new methods for automated macromolecular crystal structure solution.

CRANK is a novel suite for automated macromolecular structure solution and uses recently developed programs for substructure detection, refinement, and phasing. CRANK utilizes methods for substructure detection and phasing and combines them with existing crystallographic programs for density modification and automated model building in a convenient and easy-to-use CCP4i graphical interface. The data model used conforms to the XML eXtensible Markup Language specification and works as a common language to communicate data between many different applications inside and outside of the suite. The application of CRANK on various test cases has yielded promising results: with minimal user input, CRANK can produce better quality solutions over currently available programs.

Macromolecular crowding in biological systems: hydrodynamics and NMR methods.

Most biologically relevant environments involve highly concentrated macromolecular solutions and most biological processes involve macromolecules that diffuse and interact with other macromolecules. Macromolecular crowding is a general phenomenon that strongly affects the transport properties of macromolecules (rotational and translational diffusion) as well as the position of their equilibria. NMR methods can provide information on molecular interactions, as well as on translational and rotational diffusion. In fact, rotational diffusion, through its determinant role in NMR relaxation, places a practical limit on the systems that can be studied by NMR. While in dilute solutions of non-aggregating macromolecules this limit is set by macromolecular size, in crowded solutions excluded volume effects can have a strong effect on the observed diffusion rates. Hydrodynamic theory offers some insight into the magnitude of crowding effects on NMR observable parameters.

Supramolecular polymerization and gel formation of bis(merocyanine) dyes driven by dipolar aggregation.

Two highly dipolar merocyanine dyes were tethered by a rigid tris(n-dodecyloxy)xylylene unit that preorganizes the dyes for a supramolecular polymerization process through intermolecular aggregation of the dyes. UV/vis spectroscopy revealed a solvent dependent equilibrium between monomeric dyes and two different types of dye aggregates that are characterized by hypsochromically shifted D- and H-type absorption bands. Taking into account the ditopic nature of the supramolecular building blocks, the occurrence of the D-band indicates the formation of an oligomeric/polymeric supramolecular chain whereas the observation of the H-band suggests a higher order assembly. For the H-aggregated dyes, intrinsic viscosities exceed 0.65 L g(-1) in methylcyclohexane, values typically found for macromolecular solutions. At higher concentration, further association of these aggregates takes place by entanglement of the alkyl groups leading to a substantial increase in viscosity and gelation. Rheology studies show linear viscoelastic behavior which was attributed to the formation of an entangled dynamic network. AFM and cryo-TEM studies of the gel reveal long and stiff rod-type assemblies. X-ray diffraction studies for a solid film show columnar mesomorphism. Based on these results, a structural model is proposed in which six helically preorganized strands of the supramolecular polymer intertwine to form a rod with a diameter of about 5 nm. Within these rods all dyes are tightly aggregated in a tubular fashion giving rise to delocalized excitonic states, and the pi-conjugated tube is jacketed by the tridodecyloxy groups.

Swelling-solubility characteristics, granule size distribution and rheological behavior of banana ( Musa paradisiaca) starch

The rheological behavior at 25 and 60 °C together with the pasting properties of banana starch were examined through DSC, Brabender profile, swelling-solubility characteristics, granule size distribution and steady and oscillatory flow of pastes with 1–6% starch. Some of these properties and behaviors were also determined in cornstarch pastes for the purpose of comparison. The temperature of starch gelatinization was 75 °C and pastes were stable to thermal and mechanical treatment. Swelling and solubility were about 14% and 25 g/g, respectively. The median diameter D [ v, 0.5] of the uncooked starch was 24.31 μm, while that of cooked starch ranged from 59 to 66 μm depending on starch concentration. The pastes exhibited non-Newtonian shear thinning behavior that could be fitted to the Cross model. Under oscillatory shear, pasted exhibited a solid-like viscoelastic behavior mainly attributed to the volume fraction of swollen granules. Pastes are a mixture of swollen and partially disrupted granules suspended in a macromolecular solution (60 °C) and in a three-dimensional network (25 °C).

Hydrodynamics of dense arrangement of aggregates

The fractal aggregates in solid–liquid systems or polymer coils in macromolecular solutions overlap at sufficiently high the volume fraction in the system. In this range the correlation length diminishes as a power of the solid or polymer concentration. This alters the hydrodynamics of dense aggregated systems resulting in a power dependence expressing the permeability reduction with increasing concentration. The slope of the line depicted in a log–log plot is a function of the fractal dimension of aggregates. The analysis performed makes it possible to follow the aggregate size reduction with concentration until the state of complete breakage in which the aggregates no longer exist. For dense systems consisting of fractal aggregates of fine solids the connection is shown between the location of the line and the size of primary particles. The experimental data are quoted and analyzed to support the description.

In situ polymerization of unfluorinated and fluorinated acrylic copolymers for the conservation of stone

AbstractThe effectiveness of butyl methacrylate/ethyl acrylate–based copolymers as consolidants for stone was investigated. The penetration of polymeric consolidating products is extremely slight because of their macromolecular nature and in situ polymerization of the monomers is suggested as an alternative technique to the use of macromolecular solutions. The efficiencies of three products synthesized in situ and fluorinated by various copolymers were comparatively evaluated in terms of surface properties, water permeability, and consolidation on a selected stone substrate. Moreover, because the environmental stability of such polymeric products is one of the key factors for their extensive application for conservative treatments, the photooxidative stability of reference polymers prepared in vitro was also investigated. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3202–3213, 2004

Thermophoresis as a probe of particle–solvent interactions: The case of protein solutions

Thermophoresis, or thermal diffusion, consists in particle drift induced by thermal gradients. We discuss the physical mechanisms underlying thermophoresis in colloidal suspensions and macromolecular solutions, and present a general model suggesting thermophoretic measurements as a sensitive probe of particle–solvent interfacial properties. After reviewing recent results obtained by our group on thermal diffusion in charged colloidal systems, we discuss thermophoresis in protein solutions as a specific ‘case study’.

SOLVE and RESOLVE: automated structure solution, density modification and model building.

The software SOLVE and RESOLVE can carry out all the steps in macromolecular structure solution, from scaling and heavy-atom location through phasing, density modification and model-building in the MAD, SAD and MIR cases. SOLVE uses scoring scheme to convert the decision-making in macromolecular structure solution to an optimization problem. RESOLVE carries out the identification of NCS, density modification and automated model-building. The procedure is fully automated and can function at resolutions as low as 3 A.

Transport of Nucleosome Core Particles in Semidilute DNA Solutions

We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25–100mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the presence or absence of histone tails and is affected by the salt concentration due to the relaxation effect of counterions. NCPs placed in a network of long DNA molecules (30–50 kbp) reveal anomalous diffusion. We demonstrate that NCPs diffusion is in agreement with known particle transport in entangled macromolecular solutions as long as the histone tails are folded onto the particles. In contrast, when these tails are unfolded, the reversible adsorption of NCPs onto the DNA network has to be taken into account. This is confirmed by the fact that removal of the tails leads to reduction of the interaction between NCPs and the DNA network. The findings suggest that histone tail bridging plays an important role in chromatin dynamics.

Membrane crystallization of lysozyme: kinetic aspects

The relevant kinetic aspects related to an innovative method of biological macromolecules crystallization based on microporous hydrophobic membranes, used both as active surfaces to promote heterogeneous nucleation and as a mass-transfer apparatus to concentrate macromolecular solutions by solvent removal in vapour phase, have been evaluated. Polypropylene membranes, supplied in the form of hollow fibres, have been aligned in a versatile system, designed for an on-line spectrophotometric monitoring of hen egg white lysozyme crystallizing solutions (experimental conditions: 0.1 M NaAc/HAc Buffer pH 4.6, 0.5–5.8% wt/vol NaCl, 20°C). The turbidity measurements have been exploited in order to follow: (i) the induction time of crystallization, (ii) the early stage nucleation kinetics based on the Rayleigh scattering theory, and (iii) the crystal growth rate (coupled with data evaluated from image-analysis carried out by optical microscopy) under a model hypothesis of exponential growth of clusters. The crystals have been qualitatively assessed by an X-ray crystallographic analysis carried out at the synchrotron light laboratory ELETTRA.

Polyelectrolyte chain dimensions and concentration fluctuations near phase boundaries

We have measured the temperature (T) dependence of the correlation length (ξ) for concentration fluctuations in aqueous solutions of sodium–poly(styrene sulfonate) with a fixed level of added barium chloride salt. Apparent critical behavior is observed upon lowering the temperature to precipitation phase boundaries that complements our earlier work on salt-dependent behavior. We interpret experimental deviations from ξ−2 versus T−1 as crossover from the mean field to the Ising universality class. We also measured the radius of gyration (Rg) of labeled chains and ξ for semidilute polyelectrolyte solutions at low ionic strengths. We recovered the familiar result of ξ scaling with polymer concentration (Cp) and degree of polymerization (N), such that ξ=(73±9) N0Cp−0.48±0.03 [Å], and using SANS high concentration labeling Rg=(400±28)Cp−0.24±0.01 [Å] (for N=577) and Rg=(2.8±2.1)N0.6±0.1 [Å] (for Cp=206 gL−1), respectively. The indices recovered are in agreement with theoretical predictions for low ionic strength semidilute solutions. Such experiments offer insight into relatively unexplored phase behavior in charged macromolecular solutions.

A manual nanoscale method for protein crystallization.

To overcome one of the major hurdles in three-dimensional crystal structure determination - the requirement for large quantities of purified material to grow crystals - crystallization methodologies have been developed that require only a total of 2-5 microl of a concentrated macromolecular solution to screen more than 100 conditions. These procedures employ a circular slide containing an array of 25 wells designed for crystallization setups in the nanolitre volume range. These 'crystallization slides' fit into the wells of standard crystallization trays. These nanoscale crystallization approaches have been used to reproducibly obtain well diffracting crystals of three proteins, two that are being actively studied (glycerol kinase and NADH peroxidase) and one test protein (lysozyme), using only 40-350 microg (0.04-0.35 mg) of proteins to screen 100 conditions. These nanolitre crystallization methods are easily adapted for the typical laboratory, without the requirement of robotics or expensive equipment.

Dynamics of bound and free water in an aqueous micellar solution: Analysis of the lifetime and vibrational frequencies of hydrogen bonds at a complex interface

In order to understand the nature and dynamics of interfacial water molecules on the surface of complex systems, large scale, fully atomistic molecular dynamics simulations of an aqueous micelle of cesium perfluorooctanoate (CsPFO) surfactant molecules have been carried out. The lifetime and the intermolecular vibrational frequencies of the hydrogen bonds that the water molecules form with the hydrophilic, polar head groups (PHG) of the surfactants are calculated. Our earlier classification [S. Balasubramanian et al., Curr. Sci. 84, 428 (2003); e-print cond-mat/0212097] of the interfacial water molecules, based on structural and energetic considerations, into bound and free types is further validated by their dynamics. Lifetime correlation functions of the water-surfactant hydrogen bonds show the long-lived nature of the bound water species. Surprisingly, the water molecules that are singly hydrogen bonded to the surfactants have a longer lifetime than those that form two such hydrogen bonds. The free water molecules that do not form any such hydrogen bonds behave similarly to bulk water in their reorientational dynamics. A few water molecules that form two such hydrogen bonds are orientationally locked in for durations of the order of a few hundreds of picoseconds; that is, much longer than their average lifetime. The intermolecular vibrational frequencies of these interfacial water molecules have been studied from the power spectra of their velocity autocorrelation function. We find a significant blue shift in the librational band of the interfacial water molecules, apart from a similar shift in the near neighbor bending modes, relative to water molecules in bulk. These blue shifts suggest an increase in rigidity in the structure around interfacial water molecules. This is in good agreement with recent incoherent, inelastic neutron scattering data on macromolecular solutions [S. Ruffle et al., J. Am. Chem. Soc. 124, 565 (2002)]. The results of the present simulations appear to be rather general and should be relevant to the understanding of the dynamics of water near any hydrophilic surface.

An approximate method for calculating depletion and structural interactions between colloidal particles

An approximate method is evaluated for calculating the second virial coefficient in a dilute macromolecular solution bounded by two interfaces. The approximation is essentially the superposition of the coefficients calculated independently for each surface. To test this approach, the depletion interaction between two particles in a solution of nonadsorbing, spherical macromolecules is calculated in systems with either hard-wall or long-range electrostatic interactions. In all systems tested, the interparticle interaction energy calculated with the approximation is found to be in good agreement with that calculated using the exact approach (e.g., error less than 2% at the smallest separations). The primary advantage of this approximation is a significant reduction in the computation time required for calculating the depletion interaction, especially in charged systems. The paper also shows that the expressions for predicting the depletion interaction in purely hard-sphere systems can be used in dilute ionic systems, provided the appropriate effective macromolecule size is used. For the attractive depletion interaction, this effective size is determined by the range of the particle–macromolecule interaction (as opposed to the macromolecule–macromolecule interaction).

Ionic Conduction of Solid Polymer Electrolytes Based on Acrylonitrile–Butadiene Copolymer and Lithium Hexafluoroarsenate

The ionic structure and transport properties intrinsic to solid polymer electrolytes based on an acrylonitrile–butadiene (40 : 60) copolymer and lithium hexafluoroarsenate are studied at the salt concentrations close to the salt solubility limit in the polymer matrix. The ionic structure of a macromolecular ionic solution alters with increasing salt concentration and temperature, as does the ionic-transport mechanism. Possible models for the ionic structure of concentrated solid polymer electrolytes and ionic-transport mechanisms corresponding to them are considered.

Automated structure solution, density modification and model building.

The approaches that form the basis of automated structure solution in SOLVE and RESOLVE are described. The use of a scoring scheme to convert decision making in macromolecular structure solution to an optimization problem has proven very useful and in many cases a single clear heavy-atom solution can be obtained and used for phasing. Statistical density modification is well suited to an automated approach to structure solution because the method is relatively insensitive to choices of numbers of cycles and solvent content. The detection of non-crystallographic symmetry (NCS) in heavy-atom sites and checking of potential NCS operations against the electron-density map has proven to be a reliable method for identification of NCS in most cases. Automated model building beginning with an FFT-based search for helices and sheets has been successful in automated model building for maps with resolutions as low as 3 A. The entire process can be carried out in a fully automatic fashion in many cases.

Membrane crystallization of macromolecular solutions

Crystal growth is the critical step in determining the protein X-ray crystal structure. Hundreds, or even thousands, of crystallization trials must often be performed on a target macromolecule and, unfortunately, less than 1% of them typically yield promising results. Many macromolecules are reluctant to crystallize and, usually, their crystalline arrangement is not good enough to provide diffraction data at a resolution sufficient to establish structure-function correlation. The history of macromolecular crystallization emphasizes the importance of new observations and ideas that are useful in initiating more systematic studies using novel techniques and creative approaches. On this basis an innovative methodology, the membrane crystallization, has been introduced in order to promote the formation of macromolecular crystals. Lysozyme crystals, produced by removing the solvent (in vapour phase) from the protein solution by using microporous hydrophobic membrnaes, showed a good structural quality suitable for successive X-ray diffraction analysis.

In situ copolymerization for the consolidation of stone artefacts

In this paper, the properties of some polymeric materials used as consolidating and protective products in the restoration field of stone manufactured objects of artistic interest are discussed. The penetration of polymeric consolidating products is very small because of their macromolecular nature, and in situ polymerization of the monomers is suggested as an alternative technique to the use of macromolecular solutions. The effects of plasticizer agents in the reaction system have been investigated, in order to decrease Tg and make the products more elastic. Abrasion test.

Determination of distribution of colloidal particles on their parameters in electro-optical investigation

Abstract In this paper statistical approach is applied to electro-optical investigation of electric properties and sizing of colloidal panicles. Although the main attention is paid to colloid systems, the main principles may be extended to macromolecular solutions. Theoretical description of electro-optical effects in colloidal systems and macromolecular solutions results in Fredholm integral equations of the first kind for the particle distribution in their parameters, such as dipole moment, size, coefficient of rotary diffusion. Regularization technique is applied to solution of these equations and its feasibility is tested. The investigation of particle interaction and the determination of relations between different particle parameters can be carried out taking into consideration the distribution of particles on their parameters. The main principles of the paper are supported by experimental results.

On the role of the macromolecular phase transitions in biology in response to change in solution volume or macromolecular composition: action as an entropy buffer

We have used numerical simulation to demonstrate the potential for macromolecular precipitate solution phase transitions existing within the cell, to play a role in the minimization of changes in location or quaternary state of other macromolecular components, predicted to accompany changes in cell volume. For our modeling we have employed thermodynamic relations that take into account the large effects upon the thermodynamic activity coefficient produced by a solution environment that is highly volume occupied due to the presence of high concentrations of soluble macromolecule. The theoretical approach adopted, along with the simulated results, provide a framework for the interpretation of certain proteins’ behavior (e.g. cytoskeletal elements such as tubulin and actin and possibly some prion structures) in response to cell volume change.