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Niemann-Pick type C2 (NPC2) protein is a soluble protein that binds unesterified cholesterol. The protein helps transport unesterified cholesterol in tandem with the membrane protein Niemann-Pick type C1 (NPC1). Defects in either of proteins can cause Niemann-Pick type C disease (NPC), which results in the accumulation of unesterified cholesterol and lipids in the late endosome and lysosome. NPC is an autosomal recessive lysosomal storage disease affecting 0.35~2.20 per 100,000 people. Here we present the structural analysis of the human NPC2 glycoprotein, including expression, purification, functional analysis, homology modeling, and crystallographic studies. Human NPC2 was expressed from baculovirus-infected Trichoplusia ni (Tn5) insect cells. The construct contained a hexahistidine purification epitope tag, and the protein was purified using Nickel affinity column chromatography. The purified protein was used in binding studies with dehydroergosterol (DHE), showing that human NPC2 was functional. Using the structure of bovine NPC2, we made a homology model and mapped the human mutations onto the model. Some modeled proteins, such as the V30M and S67P variants, are unclear as to how they lead to disease, thus a structure of the human protein would be informative. Crystallization screens of human NPC2 were performed and led to crystals with a needle-like morphology, which diffracted to 4Å resolution. The structure of human NPC2 will be useful for understanding the mechanism of cholesterol binding and trafficking in cells, and to better understand the human metabolic disease NPC.

No description available.

No description available.

Conjugated semi-conducting polymers have become well known for their potential applications in hybrid electronic devices like solar cells, LEDs, and organic displays. These hybrid devices also contain inorganic nanoparticles, which complement the polymer when they are combined into the same layer. Control over the conformation and crystallinity of the polymer is critical for device performance, yet not much is known about the effect that these nanoparticles have on the polymer. Here, zinc oxide nanowire was surface modified with mono-substituted-carboxylic acid tetraphenylporphyrin and dodecanethiol, and introduced to poly(3-hexyl thiophene) in solution. The electron transfer, kinetics, and thermodynamics of this system were investigated through spectroscopic methods. Chemical reaction rate laws and Lauritzen-Hoffman Growth Theory were employed to substantiate the mechanism and rate of polymer crystallization. Surface-modification of the ZnO nanowire suggested an improvement in polymer nucleation by as much as 43.8%. A synthetic procedure was also developed to modify the inorganic nanowire with quantum dots in order to improve electron transport into the nanowire. Development of these theories and exploration of these surface effects can help lead the way for a new generation of flexible, high efficiency, hybrid electronic devices.

The main theme of this research is to understand the structure-property evolution during crystallization of a semicrystalline thermoplastic polymer. A combination of techniques including rheology, small angle light scattering, differential scanning calorimetry and optical microscopy are applied to follow the mechanical and optical properties along with crystallinity and the morphology. Isothermal crystallization experiments on isotactic poly-1-butene at early stages of spherulite growth provide quantitative information about nucleation density, volume fraction of spherulites and their crystallinity, and the mechanism of connecting into a sample spanning structure. Optical microscopy near the fluid-to-solid transition suggests that the transition, as determined by time-resolved mechanical spectroscopy, is not caused by packing/jamming of spherulites but by the formation of a percolating network structure. The effect of strain, Weissenberg number (We) and specific mechanical work (w) on rate of crystallization (nucleation followed by growth) and on growth of anisotropy was studied for shear-induced crystallization of isotactic poly-1-butene. The samples were sheared for a finite strain at the beginning of the experiment and then crystallized without further flow (Janeschitz-Kriegl protocol). Strain requirements to attain steady state/ leveling off of the rate of crystallization were found to be much larger than the strain needed to achieve steady state of flow. The large strain and We >1 criteria were also observed for morphological transition from spherulitic growth to oriented growth. An apparatus for small angle light scattering (SALS) and light transmission measurements under shear was built and tested at the University of Massachusetts Amherst. As a new development, the polarization direction can be rotated by a liquid crystal polarization rotator (LCPR) with a short response time of 20 ms. The experiments were controlled and analyzed with a LabVIEWTM based code (LabVIEWTM 7.1) in real time. The SALS apparatus was custom built for ExxonMobil Research in Clinton NJ.

<p> The opening of the Rio Grande Rift of Southern Colorado resulted in a wide variety of igneous features including the Spanish Peaks dual stocks, their surrounding radial dyke system and several proximal enigmatic plugs.</p> <p> Aspects of the crystallization history of two magmas have been studied by using petrographic observations and Crystal Size Distribution (CSD). These were used to examine opaques and feldspars from a rhyolitic dyke in the Spanish Peaks swarm and augite, olivine, and opaques from a gabbroic dyke within Huerfano Butte.</p> <p> Augite and opaques from Huerfano Butte reveal a history of no crystal fractionation during emplacement, no size dependent growth, and constant, continuous nucleation and growth.</p> <p> Huerfano Butte olivine and Spanish Peaks opaques have been subjected to resorption and depletion of nucleii. The resorbed olivine material was probably redeposited as augite. This could explain why Huerfano Butte augite has a much higher nucleation density than the other minerals.</p> <p> The phenocrystic feldspars from Spanish Peaks also have a deficit of nucleii, but they lack resorption features. Xenocrystic skeletal feldspars throughout the dyke lack a fines population and show resorption. The absence of fines is probably due to sorting of grains by density during magma convection before eruption.</p> / Thesis / Master of Science (MSc)

<p> This study is on the thermodynamics, electrical and diffusion properties of five bulk metal glasses, new materials of great importance in technology, and on a longstanding problem of the residual entropy of the glassy state. It describes (i) an investigation of spontaneous structural relaxation, (ii) discovery of memory effect, and (iii) an investigation of crystallization kinetics both isothermally and on rate heating of bulk metallic glasses by measurements of their enthalpy change with time, temperature, and annealing conditions. Furthermore, it provides a real-time electrical resistivity study of structural relaxation effects, and an electron microscopy study of the interdiffusion kinetics of atoms across a junction interface, i.e., the so-called Kirkendall effect. </p> <p> It is shown that structural relaxation occurs according to a stretched exponential kinetics and distribution of relaxation times leads to memory effect for a glass sample of complex thermal history. This mechanism is confirmed by real time electrical resistivity measurements at different temperatures and explained in terms of the Ziman model. Crystallization of ultraviscous melts occurs in several steps but the first and major step follows the Kolmogorov-Johnson-Mehl-Avrami kinetics based upon the Poisson distribution of nucleation sites. Several other processes also occur including a possible spinodal decomposition with one phase remaining in the rigid glassy state. Thermally activated interdiffusion of atoms across a junction interface is inconsistent with the vacancy diffusion model. Finally, it is shown that contrary to the recent arguments based upon the Boltzmann equation, a glass has residual entropy. </p> <p> Seven papers based upon this study have been published in Journal of Non-Crystalline Solids, Journal of Chemical Physics, Journal of Physical Chemistry, Thermochimica Acta, and Philosophical Magazine. </p> / Thesis / Doctor of Philosophy (PhD)

X-ray crystallography is a popular method for resolving protein structures. Protein crystals need to be used for X-ray crystallography, but most naturally occurring proteins do not readily crystallize. The Hunt lab performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization. This stands in direct contrast to earlier work and dogmas that posited that surface entropy reduction was the clear path forward to crystallzing proteins. Arginine is a high-entropy sidechain, yet it helps drive protein crystallization. To understand which structure and dynamical features of arginine give rise to crystal packing propensity, we performed 60 Molecular Dynamics (MD) simulations to measure the sidechain order parameter of arginine and compare it against crystal packing propensity. This work found that surface-exposed arginines with low order parameters are most likely to participate in crystal packing interactions. This is evidence against earlier thinking that high entropy surface sidechains oppose crystallization. Entropic barriers to protein crystallization can be enthalpically overcome.

In this work, crystallization experiments were carried out on four separate aqueous solutions of adipic acid, ammonium sulfate, urea and L-glutamic acid to measure the growth rate of these crystals under varying values of temperature, stirrer speed, cooling rate and holding time. All experiments were carried out in the Mettler Toledo LabMax, which is an automated laboratory reactor. A polarized light microscope was used to capture the images of the crystals and Image Pro Plus software was used for the analysis of crystal samples. Due to technical difficulties, the data could not be measured for adipic acid, ammonium sulfate or urea. L-Glutamic acid was much easier to work with and it was possible to obtain data. The growth rate for the b form of L-glutamic acid was estimated from the experimental data using a numerical simulation