Changes In Salt Concentration Research Articles

Engineering proteins with tunable thermodynamic and kinetic stabilities

It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins.

On the Modeling of Polyelectrolyte Gels

AbstractSummary: Ionic polymer gels are very attractive actuation materials with a great similarity to biological contractile tissues. They consist of a polymer network with bound charged groups and a liquid phase with mobile ions. Absorption and delivery of solvent lead to a considerably large change of volume. This swelling mechanism results from the equilibrium of different forces such as osmotic pressure forces, electrostatic forces and viscoelastic restoring forces and can be triggered by chemical (change of salt concentration or pH in the solution), thermal or electrical stimulation. In the present work, chemically and electrically stimulated electrolyte polymer gels in a solution bath are investigated. To describe the different phenomena occurring in these gels adequately, the modeling can be conducted on different scales. If only the global macroscopic behavior is of interest, the statistical theory which is capable to describe the global swelling ratio, is sufficient. By refining the scale, the mesoscopic coupled multi‐field theory can be applied. Here, the chemical field is described by a convection‐diffusion equation for the different mobile species. The electric field is directly obtained by solving the Poisson equation in the gel and solution domain. The mechanical field is formulated by the momentum equation. By further refining the scale, the whole structure can be investigated on the microscale by the discrete element (DE) method. In this model, the material is represented by distributed particles comprising a certain amount of mass; the particles interact with each other mechanically by a truss or beam network of massless elements. The mechanical behavior, i.e. the dynamics of the system, is followed by solving the Newton's equations of motion while the chemical field, i.e. the ion movement inside the gel and from the gel to the solution, is described by diffusion equations for the different mobile particles. All three formulations can give chemical, (electrical) and mechanical unknowns and all rely on the assumption that the concentration differences between the different regions of the gel and between gel and solution form the osmotic pressure difference, which is a main cause for the mechanical deformation of the polyelectrolyte gel film.

On the Modeling of Polyelectrolyte Gels

Summary: Ionic polymer gels are very attractive actuation materials with a great similarity to biological contractile tissues. They consist of a polymer network with bound charged groups and a liquid phase with mobile ions. Absorption and delivery of solvent lead to a considerably large change of volume. This swelling mechanism results from the equilibrium of different forces such as osmotic pressure forces, electrostatic forces and viscoelastic restoring forces and can be triggered by chemical (change of salt concentration or pH in the solution), thermal or electrical stimulation. In the present work, chemically and electrically stimulated electrolyte polymer gels in a solution bath are investigated. To describe the different phenomena occurring in these gels adequately, the modeling can be conducted on different scales. If only the global macroscopic behavior is of interest, the statistical theory which is capable to describe the global swelling ratio, is sufficient. By refining the scale, the mesoscopic coupled multi-field theory can be applied. Here, the chemical field is described by a convection-diffusion equation for the different mobile species. The electric field is directly obtained by solving the Poisson equation in the gel and solution domain. The mechanical field is formulated by the momentum equation. By further refining the scale, the whole structure can be investigated on the microscale by the discrete element (DE) method. In this model, the material is represented by distributed particles comprising a certain amount of mass; the particles interact with each other mechanically by a truss or beam network of massless elements. The mechanical behavior, i.e. the dynamics of the system, is followed by solving the Newton's equations of motion while the chemical field, i.e. the ion movement inside the gel and from the gel to the solution, is described by diffusion equations for the different mobile particles. All three formulations can give chemical, (electrical) and mechanical unknowns and all rely on the assumption that the concentration differences between the different regions of the gel and between gel and solution form the osmotic pressure difference, which is a main cause for the mechanical deformation of the polyelectrolyte gel film.

Salt-concentration dependence of the structure and form factors for the wormlike micelle made from a dual surfactant in aqueous solutions

Small-angle X-ray scattering (SAXS) from dual-surfactant aqueous solutions made from sodium lauryl ether sulfate and coconut fatty acid amido propyl betaine was systematically measured as a function of the net sodium cation concentration, [Na+]*, and the surfactant concentration, CD. The SAXS intensity [I(q)] was normalized to CD and the resultant I(q)/CD was extrapolated to CD = 0 to give a form factor P(q) for each [Na+]* [where q = 4πsin(θ/2)/λ is the magnitude of the scattering vector, λ is the wavelength and 2θ is the scattering angle]. The low-q behaviour of P(q) was consistent with long rigid cylinders. The middle- and high-q profiles fitted well with a core–shell cylinder model for all [Na+]*. The core and total radii (Rc and Rs) did not depend on [Na+]* at all: Rc = 1.2 ± 0.05 and Rs = 3.1 ± 0.05 nm for [Na+]* = 0.42–1.5 mol l−1, indicating that the salt concentration changes did not induce any structural changes and re-assembling of the surfactants comprising the micelles. This fact is in contrast to the rheological behaviour where the relaxation mode strongly depends on [Na+]*. The structure factor [S(q)] was obtained by dividing I(q)/CD by P(q) for each CD and the mean distance (dm) between the micelles was obtained from the first maximum of S(q) versus q plots. The dm value decreased with increasing CD and [Na+]*, which is in good agreement with the theoretical prediction and experimental results for charged wormlike micelle solutions.

Studies of electrochemical properties of compacted clays by concentration potential method

The development of concentration (membrane) potential upon step-wise change in salt concentration has been studied for diaphragms made of various strongly compacted clays (montmorillonite, illite, kaolinite) equilibrated with 0.1 M NaCl solution. Porous ceramic filters were used to confine the clays mechanically to be able to achieve high extent of compaction (dry density ∼2000 kg/m 3). A theoretical analysis has revealed that the relaxation pattern is primarily controlled by the properties of porous filters and only slightly depends on the clay properties. At the same time, quasi-stationary values of concentration potential are directly related to the electrochemical perm-selectivity of clay. This property has revealed considerable differences in the electrochemical behaviour of various clays used in this study. This has been attributed to the differences in the micro-structure of clays, in particular to the existence or nonexistence of the so-called interlayer water where cations may retain some mobility. It has also been shown that in clays with high electrochemical perm-selectivity, one can expect a strong increase in the diffusivity of cationic radio-tracers with decreasing ionic strength of equilibrium electrolyte solution. At the same time, low electrochemical perm-selectivity means no noticeable dependence of this kind. The correctness of this observation has been corroborated by the comparison of our findings with the literature data on the diffusion of cationic radio-tracers through compacted montmorillonite (high perm-selectivity) and kaolinite (low perm-selectivity). To check the self-consistency of our approach, we have also carried out sample measurements of diffusion of cationic and anionic radio-tracers through compacted illite. It has been found that the measured effective diffusion coefficients were in excellent agreement with the electrochemical perm-selectivity estimated for this clay from the measurements of concentration potential.

Multipattern Rietveld refinement of protein powder data: an approach to higher resolution

By using combinations of multiple protein powder diffraction patterns obtained from an image plate to ad-spacing of 2 Å, which differ by solvent-induced and radiation-damage-induced lattice strains, the powder overlap problem is partially resolved in a stereochemically restrained Rietveld refinement. The results for hen egg white lysozyme (HEWL) include, for the first time with powder data, placement of a substantial number of water molecules and structural results that approach the quality normally obtained by single-crystal methods. This study explores the lattice strains induced by changes in salt concentration, changes in solvent pH and the effect of low-dose radiation damage. For HEWL, lattice strains are not monotonic, so that with increasing NaCl concentration (0.25–1.25 M) theaaxis increases by ∼0.5%, while thecaxis decreases by ∼1.5%, and this variation is pH dependent. Low-dose radiation damage similarly induces non-monotonic lattice strains, similar but smaller than those arising from increased salt concentration. The effect of using these powder data in a combined Rietveld analysis is effectively to deconvolute the overlapping reflections by differing shifts in their relative positions.

Protein-Based Hydrogels for Cell Transplantation under Constant Physiological Conditions

ABSTRACTA promising treatment for multiple neurological disorders including stroke, Huntington's, and Parkinson's is the transplantation of stem cells into the diseased site to promote regeneration of the neural tissue. However, viability of transplanted cells is often low (15-35%) and unpredictable. Cell viability has been directly correlated with functional outcome of the treatment, motivating the development of more reliable cell transplantation procedures. To protect transplanted cells from shear stress during injection and from the hostile, inflammatory environment of the diseased brain tissue, many research groups are exploring physical hydrogels as a protective, growth-permissive matrix to enhance cell viability. However, physical hydrogels require an environmental trigger to induce gelation. These environmental triggers include sudden changes in temperature, pH, or salt concentration - all of which are detrimental to encapsulated cells and proteins and complicate their use in a clinical environment. To address this need, we have designed a two-component, protein-based hydrogel system that can self-assemble under constant physiological conditions.Both components of the hydrogel system are created using recombinant protein technology, which allows synthesis of exact monomer sequences within monodisperse polymers. The first hydrogel component is a block copolymer made of several repeats of a peptide sequence encoding the WW domain-fold, a short triple-stranded, anti-parallel, beta-sheet. The WW domains are interspersed with a random-coil, hydrophilic spacer to enhance polymer flexibility and solubility. The second hydrogel component is made of several repeats of a polyproline rich peptide sequence interspersed with a random-coil, hydrophilic spacer. Upon mixing the two hydrogel components together, the WW-domains in component 1 and the polyproline rich peptides in component 2 bind together with 1:1 stoichiometry. This binding has an apparent association constant of 2.2×105 M, as measured by isothermal titration calorimetry. This peptide-binding event serves as the physical crosslinks to form a polymeric network composed of the two components. Because gelation is initiated by simply mixing the two components together at physiological pH, temperature, and ionic strength, this system is highly biocompatible and easy to use. Furthermore, the precision of protein engineering allows both components to be easily modified. For example, increasing the length of the hydrophilic spacers will increase the resulting network pore size. Additionally, bioactive peptide sequences, such as the RGD cell-binding domain, have been introduced into the hydrophilic spacers to modify cell-scaffold interactions. Our long-term objective is to design a self-assembling hydrogel system for cell delivery that will both improve cell viability and mimic many of the essential cues in the developmental niche to encourage cell differentiation and outgrowth.

Molecular dynamics simulation of the polymer electrolyte poly(ethylene oxide)/LiClO4. II. Dynamical properties

The dynamical properties of the polymer electrolyte poly(ethylene oxide) (PEO)LiClO(4) have been investigated by molecular dynamics simulations. The effect of changing salt concentration and temperature was evaluated on several time correlation functions. Ionic displacements projected on different directions reveal anisotropy in short-time (rattling) and long-time (diffusive) dynamics of Li(+) cations. It is shown that ionic mobility is coupled to the segmental motion of the polymeric chain. Structural relaxation is probed by the intermediate scattering function F(k,t) at several wave vectors. Good agreement was found between calculated and experimental F(k,t) for pure PEO. A remarkable slowing down of polymer relaxation is observed upon addition of the salt. The ionic conductivity estimated by the Nernst-Einstein equation is approximately ten times higher than the actual conductivity calculated by the time correlation function of charge current.

Polyelectrolyte Complexes with pH-Tunable Solubility

Polyelectrolyte complexes (PECs) with pH-tunable solubility were formed from a random copolymer of diallyldimethylammonium (DADMA) and acrylic acid (AA) having a net positive charge as polycation and poly(styrenesulfonate) as polyanion. Quasi-soluble PEC nanoparticles could be produced from solutions of exceptionally high polymer concentration by manipulating pH and ionic strength. The aggregation of the PEC nanoparticles, reversible using either changes in salt concentration or pH, exhibited well-defined hysteresis. Dynamic and static light scattering as well as transmission electron microscopy showed well-defined particles produced in the quasi-soluble limit. Viscosity and titration experiments supported a core−shell model for the dispersed nanoparticles and revealed preferential ion pairing of DADMA with sulfonic acid groups over AA units.

PH transitions in cation exchange chromatographic columns containing weak acid groups

Complex pH transitions occur in cation exchange columns used for protein chromatography during equilibration and salt elution steps when the stationary phase contains weak acid groups even if the mobile phase is buffered and the buffering species do not interact with the stationary phase. In this work, we present a local equilibrium model to predict the magnitude and duration of these pH transients. The model equations are solved by the method of characteristics and by numerical simulations using an equilibrium-dispersive model. By incorporating an explicit description of the dissociation of the weakly ionogenic groups in the resin, we show that counterion binding in the column can be predicted for different buffer systems based on a single experimental resin titration curve without having to resort to empirically defined adsorption equilibrium constants. Model predictions based on these assumptions are found to be in excellent agreement with experimental results obtained for three different resins containing varying concentrations of weak acid groups. Four common buffer systems, acetate, citrate, MES, and phosphate are considered with both step and gradient changes in salt concentration at pH 5.5. Each buffer yields a different pH excursion behavior. We demonstrate that when the counterion concentration is kept constant in each of these buffers, which is needed to attain identical protein adsorption behavior, the magnitude of the pH transitions occurring during salt steps is nearly independent of the buffer system. On the other hand, the duration of the pH transitions is smallest for MES suggesting that this buffer system is preferable where pH variations are to be prevented.

Cation binding linked to a sequence-specific CAP–DNA interaction

The equilibrium association constant observed for many DNA–protein interactions in vitro ( K obs) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of log K obs versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270–370, is determined to 1.75Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

Transient rheology of solvent-responsive complex fluids by integrating microrheology and microfluidics

A new microrheology set-up which allows us to quantitatively measure the transient rheological properties of solvent-responsive complex fluids was constructed by integrating particle tracking microrheology and microfluidics. The dialysis cell consists of a reservoir, porous dialysis membrane, and sample chamber. Solvent molecules can freely diffuse between the reservoir and the sample chamber while macromolecular sample components are trapped in the sample chamber with a rigid semipermeable dialysis membrane. The design enables manipulation of the solvent composition in the sample chamber by simply switching the fluid composition in the reservoir. Validation experiments for solvent diffusion in the dialysis cell showed good agreement with numerical solutions of the transport equations and confirmed that the solvent composition in a sample can be changed in a controlled and predictable fashion within a few minutes due to the small device dimensions. For aqueous solutions of sodium alginate and sodium polystyrene sulfonate, transient rheological properties were measured in response to changes in salt concentration. The results showed that the dialysis cell is a useful and versatile tool to study the response of complex fluids to reversible changes in solvent composition.

Osmotically unresponsive water fraction on proteins: Non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration

How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.

Single Component Chitosan Hydrogel Microcapsule from a Layer-by-Layer Approach

Smart biomaterials have found broad applications in modern medical technologies.1 However, the development of smart hydrogels with rapid response, good biocompatibility, and controllable degradability is still a formidable challenge. Chitosan, a biodegradable, nontoxic, and renewable linear polysaccharide, has been considered for various biomedical and pharmaceutical applications.2,3 To these ends, microparticles of chitosan in hundreds of micrometers have been prepared in different ways, including coacervation/ precipitation, spray-drying, emulsion cross-linking, emulsiondroplet coalescence, reverse micellization, ionic gelation, and sieving method.3,4 Complexation between chitosan and oppositely charged polysaccharides in solution is another way to synthesize chitosan microparticles; however, this process often leads to the formation of fibers.5 A new strategy, the so-called “layer-by-layer (LbL) assembly”, has been used here to fabricate microcapsules from chitosan. This strategy, based on the electrostatic interaction between oppositely charged polyelectrolytes, was first introduced by Decher et al. to fabricate thin films on planar supports by alternate adsorption of polycation and polyanion.6 Because of its advantages of fine control of the compositions and the thickness of the capsules, the LbL method received more and more attention in recent years.7-9 It was extended to three-dimensional (3D) systems by Mohwald et al. to fabricate core-shell particles and hollow capsules by further removal of the sacrificial core.10 In previous works, we first extended the LbL method based on hydrogen and covalent bonding to 3D systems and synthesized hydrogen or covalent bonded capsules.11,12 Chitosan bears positive charges and is a polycation at low pH. It has been used widely to fabricate LbL films13-19 and capsules20-22 with other polyelectrolytes. For the normal LbL methods, the capsule wall has at least two compositions. In certain applications, one may take advantage of the feasibility of several components by combining their individual material properties. On the other hand, the presence of a second polymer component for LbL assembling may limit the specific functionality of materials. For instance, the biocompatibility and biodegradability of chitosan could be destroyed by a second polymer component. Moya et al. fabricated a nearly single component capsule of polyallylamine hydrochloride (PAH) from the polystyrene sulfonate (PSS)/PAH LbL assembly by using cells as the template.23 The PAH in the capsule wall was oxidized and cross-linked, and then most of PSS was removed while removing the cell core template with sodium hypochlorite. However, this strategy could not be a general method for fabrication of single component capsules. Zhang et al. reported the preparation of single component 2-dimensional films from hydrogen-bonded LbL films, but these films were not cross-linked.24,25 Our new strategy is to selectively crosslink the chitosan chains and remove the second polymer component that was built up in the capsule walls (see Scheme 1). Using this strategy, single component chitosan hydrogel microcapsules were successfully synthesized. The pHresponsive volume phase transition of the chitosan hydrogel microcapsules could be controlled through the cross-linking density. The resultant chitosan gel microcapsules are nearly monodispersed, and the shell thickness is tunable, which is a big advantage to control the releasing kinetics of encapsulated drug or other molecules in response to pH or salt concentration changes. More importantly, although many shell cross-linked capsules have been made from synthetic block copolymers through a strategy of self-assembly of block copolymers with selective cross-linking on the shell block,26-31 it is very challenging, if not impossible, to synthesize well-defined block copolymers from natural polysaccharides. Our novel strategy here can provide a general method to synthesize shell cross-linked microcapsules from natural polysaccharides that have many applications in the pharmaceutical and medical field due to their biocompatibility and degradability.

Development of a Monoclonal Antibody-Based Enzyme-Linked Immuosorbent Assay for the β-Adrenergic Agonist Zilpaterol

Zilpaterol is a beta-adrenergic agonist approved for use as a growth promoter in cattle in South Africa and Mexico but not in the European Union, United States, or Asia. Here, we report the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for zilpaterol. Mice immunized with zilpaterol-butyrate-keyhole limpet hemocyanin were utilized for monoclonal antibody generation whereas zilpaterol-butyrate-bovine serum albumin was used as a coating antigen for ELISA. Thirteen clones were isolated, and after the initial sensitivity and isotyping experiments, three clones were selected for further ELISA optimization. Studies indicated that the optimum pH was near 7.4. Clone 3H5 had the highest sensitivity to zilpaterol and some interaction with clenbuterol and terbutaline at high concentrations but not other N-alkyl [bamethane, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists tested. However, clone 3H5 was not functional at high salt concentrations, which precluded further development for urine analysis. Clone 2E10 showed increased sensitivity as salt concentrations were increased and did not cross-react with any of the structural analogues tested. However, its sensitivity to salt and urine concentration changes could cause high variability. Clone 7A8 showed good sensitivity and only a modest change with the salt concentration changes. Clone 7A8 also demonstrated smaller changes in IC(50) and B(0) with increasing sheep urine or cattle urine concentrations as compared to clones 2E10 or 3H5 and, thus, was selected for further development. The IC(50) for all of the antibodies showed exponential increases with increasing organic solvents concentrations, making it desirable to minimize solvent levels. In conclusion, a sensitive, specific zilpaterol monoclonal antibody-based ELISA has been developed that can serve as a rapid screening assay.

Study of rice-starch structure by dynamic light scattering in aqueous solution

The expansion of various rice starches in solution was studied using dynamic light scattering (DLS) in order to provide information on their microstructure. Hydrodynamic diameters of starch molecules were found to change with solvent conditions. The component starches in the rice varieties studied had similar sizes in pure water (between 125 and 235 nm), but showed different expansion behaviour with changes in salt concentration, with addition of urea (which disrupts hydrogen bonding) and with the addition of 1-butanol (which reduces solvent polarity). Some starches showed a gradually increasing size with increasing [NaCl], while others showed an initial steep increase followed by more gradual behaviour. The size distributions from DLS indicated two (and possibly three) components at about 100 and 1000 nm. The larger component was largely responsible for the expansion with added salt, an effect which is ascribed to the greater ability of larger chains to expand. Chain length distributions (CLD) of debranched starch from the same samples were examined by capillary electrophoresis (CE) for correlation with the expansion behaviour obtained by DLS. The ratio of shorter to longer chains partly correlated with the classes of expansion behaviour. The data suggest that the expansion behaviour is sensitive to the branching structure (connectivity) as well as to the distribution of the lengths of the branches.

Chemosensory behavior of semi-restrainedCaenorhabditis elegans

A new behavioral assay is described for studying chemosensation in the nematode Caenorhabditis elegans. This assay presents three main characteristics: (1) the worm is restrained by gluing, preserving correlates of identifiable behaviors; (2) the amplitude and time course of the stimulus are controlled by the experimenter; and (3) the behavior is recorded quantitatively. We show that restrained C. elegans display behaviors comparable to those of freely moving worms. Moreover, the chemosensory response of wild-type glued animals to changes in salt concentration is similar to that of freely moving animals. This glued-worm assay was used to reveal new chemosensory deficits of the potassium channel mutant egl-2. We conclude that the glued worm assay can be used to study the chemosensory regulation of C. elegans behavior and how it is affected by neuronal or genetic manipulations.

Physical Detwinning of Hemihedrally Twinned Hexagonal Crystals of Bacteriorhodopsin

Hexagonal crystals of the membrane protein bacteriorhodopsin of space group P6 3 grown in lipidic cubic phase are twinned hemihedrally. It was shown that slow changes of salt concentration in the mother liquor lead to a split of crystals so that the split parts preserved high diffraction quality. Analysis of diffraction data from split crystals by Yeates statistic and Britton plot showed that the split parts are free of twinning. It is concluded that crystals of bacteriorhodopsin are composed of several macroscopic twinning domains with sizes comparable to the original crystal. The appearance of twinning domains during crystal growth and the mechanism of splitting are discussed.

Novel role of tyrosine in catalysis by human AP endonuclease 1

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5′ to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr 128 and Tyr 269, which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr 171 is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000–50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr 171 mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr 171 is the nucleophile that attacks the scissile phosphate.