Biomedical subjects

Charles F Zukoski

Publications and source records attributed to Charles F Zukoski.

11 recordsLinked to original sources

PubMed2006

Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase.

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.

Binding Sites↗

PubMed2006

Aggregation and gelation kinetics of fumed silica-ethanol suspensions.

The kinetics of aggregation and gelation of fumed silica suspended in ethanol were investigated as a function of volume fraction. At low particle concentrations, gelation is well described by aggregation into a primary minimum arising from hydrogen bonding and dispersion forces. The gelation is extremely slow due to an energetic barrier (approximately 25 kT) in the interparticle potential associated with solvation forces. The solvation forces also contribute to the formation of a secondary minimum in the interparticle potential. The depth of this minimum (approximately 3 kT) is sufficient that, at a critical particle concentration, long-range diffusion is arrested due to the short-range attractions and the cooperative nature of particle interactions, as described by mode coupling theory. The presence of the secondary minimum is also observed in the microstructure of the gels studied using X-ray scattering. These observations reinforce the importance of understanding the role of solvent-particle interactions in manipulating suspension properties.

Journal Article↗

PubMed2006

Role of solvation forces in the gelation of fumed silica-alcohol suspensions.

Aggregation and gelation kinetics of fumed silica were investigated by altering the solvent-surface interactions. Native and surface-modified (hydrophobic) fumed silica particles were dispersed in short-chain linear alcohols. Based on the kinetics of aggregation and gelation, we show that the solvent-surface interactions have a tremendous impact on the bulk suspension properties. The gelation kinetics were qualitatively similar in all of the fumed silica-alcohol samples, and the gel times for all the alcohols were captured on a master curve requiring two parameters. The two parameters, the stability ratio and critical volume fraction, describe the two regimes of gelation. At low concentrations, gelation occurs due to aggregation of the particles diffusing over a potential barrier (15-25 kT). The rate of aggregation and time to gelation then scales with the stability ratio. At high particle loadings, gelation occurs at a critical volume fraction due to localization in a secondary minimum with a depth of 3-4 kT. These observations are supported by evidence of hydrogen bonding between the solvent and the particle, creating oscillatory solvation forces that govern the magnitude of these two parameters.

Alcohols↗

PubMed2006

Synthesis and aggregation behavior of thermally responsive star polymers.

To mimic the three-dimensional (3-D) globular architecture resulting from the precise positioning of hydrophobic/hydrophilic domains (blocks) of naturally occurring proteins, water-soluble linear and star homopolymers of N,N'-dimethylacrylamide (DMA) were synthesized with prescribed molecular weights via reversible addition-fragmentation chain transfer (RAFT) polymerization and subsequently used as macro chain transfer agents for block copolymerization with N-isopropylacrylamide (NIPAM). For the star block copolymers, the interior block consisted of NIPAM while the exterior block was DMA. Since polyNIPAM thermally switches from hydrophilic to hydrophobic, the 3-D solution conformations of the polymers were studied as a function of temperature using differential scanning calorimetry (DSC), static light scattering (SLS), and dynamic light scattering (DLS). The polymers were observed to form monodisperse aggregates in an aqueous pH 4 buffer solution when heated above the lower critical solution temperature (LCST) of polyNIPAM. The temperature at which the polymers aggregated and the size of the aggregates were dependent on the NIPAM block length and the core architecture. A simple model based on an optimal area per headgroup was used to analyze our experimental findings and was useful for predicting the final size and molecular weight of the aggregates formed.

Journal Article↗

PubMed2006

Synthesis of anisotropic nanoparticles by seeded emulsion polymerization.

Anisotropic polystyrene nanoparticles of diameters below 0.5 microm were prepared by coating the surface of cross-linked polystyrene latex particles with a thin hydrophilic polymer layer prior to swelling the particles with styrene and then initiating second-stage free-radical polymerization. Conditions were found so that all particles had uniform asymmetry. The effect of surface chemistry on the development of particle anisotropy during seeded emulsion polymerization of sub-0.5 microm diameter particles was studied. The extent and uniformity of the anisotropy of the final particles depended strongly on the presence of the hydrophilic surface coating. Systematic variation of the degree of hydrophilicity of the surface coating provided qualitative insight into the mechanism responsible for anisotropy. Conditions were chosen so that the surface free energy favored the extrusion of a hydrophobic bulge of monomer on the hydrophilic surface of the particle during the swelling phase: the presence of a hydrophilic layer on the particle surface causes this asymmetry to be favored above uniform wetting of the particle surface by the monomer. Kinetic effects, arising from the finite time required for the seed to swell with the monomer, also play a role.

Journal Article↗

PubMed2004

Hard structured particles: suspension synthesis, characterization, and compressibility.

Hard interactions are developed on three grades of fumed silica by eliminating interparticle forces and sterically stabilizing the particles by attaching an organic coating to the surface of the particles, suspending them in an index-matching solvent and screening the electrostatics. These hard-structured particles are studied to understand the effects of the particle's microstructure on suspension properties without the influence of interparticle forces other than volume exclusion, Brownian, and hydrodynamic interactions. Light and X-ray scattering studies of low-volume-fraction suspensions suggest that the fumed silicas consist of primary particle of radius of gyration R(g1) approximately equals 16 nm and aggregate size R(g2) approximately 50 nm and mass fractal dimension D(f) approximately equals 2.2. Osmotic compressibilities of these suspensions are measured as a function of particle concentration exploring the packing mechanism of fumed silica. While there is minimal detectable change in the primary particle size, R(g2) varies by approximately 15%, providing insight into how suspension properties are related to particle size. As expected of hard particles with the same microstructure, the concentration dependence on the osmotic pressure superimposes with volume fraction of solids. The comparison of fumed-silica-suspension measurements to the known behavior of hard-sphere suspensions demonstrates the effects of particle geometry on suspension properties with indications of interpenetration of the fumed silica due to their open geometry.

Journal Article↗

PubMed2004

Cold-stable eye lens crystallins of the Antarctic nototheniid toothfish Dissostichus mawsoni Norman.

The eye lenses of the Antarctic nototheniid fishes that inhabit the perennially freezing Antarctic seawater are transparent at -2 degrees C, whereas the cold-sensitive mammalian and tropical fish lenses display cold-induced cataract at 20 degrees C and 7 degrees C, respectively. No cold-cataract occurs in the giant Antarctic toothfish Dissostichus mawsoni lens when cooled to temperatures as low as -12 degrees C, indicating highly cold-stable lens proteins. To investigate this cold stability, we characterised the lens crystallin proteins of the Antarctic toothfish, in parallel with those of the sub-tropical bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, representing three disparate thermal climes (-2 degrees C, 18 degrees C and 37 degrees C, respectively). Sizing chromatography resolved their lens crystallins into three groups, alpha/betaH, beta and gamma, with gamma crystallins being the most abundant (>40%) lens proteins in fish, in contrast to the cow lens where they comprise only 19%. The upper thermal stability of these crystallin components correlated with the body temperature of the species. In vitro chaperone assays showed that fish alpha crystallin can protect same-species gamma crystallins from heat denaturation, as well as lysozyme from DTT-induced unfolding, and therefore are small Heat Shock Proteins (sHSP) like their mammalian counterparts. Dynamic light scattering measured an increase in size of alphagamma crystallin mixtures upon heating, which supports formation of the alphagamma complex as an integral part of the chaperone process. Surprisingly, in cross-species chaperone assays, tuna alpha crystallins only partly protected toothfish gamma crystallins, while cow alpha crystallins completely failed to protect, indicating partial and no alphagamma interaction, respectively. Toothfish gamma was likely to be the component that failed to interact, as the supernatant from a cow alpha plus toothfish gamma incubation could chaperone cow gamma crystallins in a subsequent heat incubation, indicating the presence of uncomplexed cow alpha. This suggests that the inability of toothfish gamma crystallins to fully complex with tuna alpha, and not at all with the cow alpha crystallins, may have its basis in adaptive changes in the protein that relate to the extreme cold-stability of the toothfish lens.

Acclimatization↗

PubMed2003

Competition between crystallization and gelation: a local description.

We develop a model to describe the competition between gelation and crystallization in colloidal suspensions where particle interactions are represented by square well attractions. The competition is discussed locally in terms of the tendencies of individual particles to attain amorphous or crystalline configurations on cluster surfaces. These tendencies are dictated by three independent processes, the aggregation of particles onto, the dissociation of particles from, and the rearrangement of particles on the cluster surfaces. Models are developed to determine the rates of each of these processes. The relative magnitudes of these rates determine the probability that a particle arriving onto a cluster surface reaches a crystalline configuration, remains arrested in an amorphous configuration, or dissociates back into the suspension. These probabilities are employed to determine whether stable crystalline or amorphous clusters nucleate, resulting in predictions of the occurrence of crystallization or gelation as a function of solution conditions. Comparisons of model predictions with recent experiments on globular protein suspensions show excellent agreement, suggesting that the model captures much of the underlying physics of the competition between gelation and crystallization in attractive colloidal suspensions.

Journal Article↗

PubMed2003

PAA/PEO comb polymer effects on rheological properties and inter-particle forces in aqueous silica suspensions.

The effects of a poly(acrylic acid) (PAA)-poly(ethylene) (PEO) comb polymer dispersant on the rheological properties and inter-particle forces in aqueous silica suspensions have been studied under varying pH conditions. The comb polymer was found to adsorb more strongly under acidic than basic conditions, indicating that the PAA backbone of the copolymer preferentially adsorbs onto silica surfaces with the PEO "teeth" extending out from the surface into the solution. In the presence of low concentrations of copolymer, the silica suspensions were stable due to electrostatic repulsions between the silica surfaces. At higher copolymer concentrations and under neutral and basic conditions, where the copolymer interacted only weakly with silica, the suspensions showed a transition from a dispersed to weakly flocculated state and attractive forces were measured between silica surfaces. Under acidic conditions, the silica dispersion also destabilized at intermediate copolymer adsorbed density and then was re-stabilized at higher adsorbed coverage. The silica suspensions were stable at high copolymer coverage due to steric repulsions between the particles. The destabilization at intermediate coverage is thought to be due to polymer bridging between particles or possibly depletion forces.

Journal Article↗

PubMed2003

Ergodic and non-ergodic phase transitions in globular protein suspensions.

The equilibrium and nonequilibrium phase behavior of a protein suspension is investigated as a function of strength of interparticle attraction and protein concentration. The equilibrium phase behavior suggests that the range of the particle attractions is a small fraction of their diameter. At volume fractions and strengths of attractions smaller than those characterizing the spinodal of a metastable fluid-fluid transition, the suspensions gel before they crystallize. At higher volume fractions and lower strengths of attraction, gels are formed first from which crystals nucleate over a period of time. However, at higher strength of attraction, crystals form first at lower volume fractions while gels are observed at higher volume fractions. We hypothesize that this behavior results from the competition between the rates of gelation and crystal nucleation. The location of the gel line is well predicted by mode coupling theories adapted for low volume fractions and square well fluids. At low strength of attraction, the gel line occurs at small supersaturations suggesting gels will be seen before nucleation. However, at higher strengths of attraction the gel line occurs at greatly increased supersaturations such that crystal nucleation can occur before the gel line is crossed. That mode coupling theories should predict gelation when crystallization does not intervene is tested by investigating the gel dynamics.

Algorithms↗

PubMed2002

Nucleation rates and induction times during colloidal crystallization: links between models and experiments.

A kinetic model for the evolution of the cluster size distribution during crystal nucleation and growth is presented. The model allows one to establish precise links between model parameters and experimental measures of nucleation kinetics. This approach demonstrates the significance of several processes not accounted for in classical nucleation theories. Chief among these is that the driving force for crystal nucleation decreases rapidly due to a reduction of the background monomer concentration as crystallization progresses, resulting in a reduction of nucleation rates. This, coupled with the disparities in the definitions of measured and predicted quantities, leads to significant discrepancies between predictions of extant models and experimental estimates of nucleation rates. Accounting for these effects, calculations of the kinetic model are shown to be in good agreement with experimental estimates of nucleation rates, crystal growth velocities, and induction times during the crystallization of hard sphere colloidal suspensions.

Journal Article↗