Engineering the nanoparticle - protein association offers a new way to form protein crystals as well as new approaches for arrangement of nanoparticles. Central to this control is the nanoparticle surface. By conjugating polymers on the surface with controlled molecular weights many properties of the nanoparticle can be changed including its size, stability in buffers and the association of proteins with its surface. Large molecular weight poly(ethylene glycol) (PEG) coatings allow for weak associations between proteins and nanoparticles. These interactions can lead to changes in how proteins crystallize. In particular, they decrease the time to nucleation and expand the range of conditions over which protein crystals form. Interestingly, when PEG chain lengths are too short then protein association is minimized and these effects are not observed. One important feature of protein crystals nucleated with nanoparticles is that the nanoparticles are incorporated into the crystals. What results are nanoparticles placed at well-defined distances in composite protein-nanoparticle crystals. Crystals on the size scale of 10 - 100 micrometers exhibit optical absorbance, fluorescence and super paramagnetic behavior derivative from the incorporated nanomaterials. The arrangement of nanoparticles into three dimensional arrays also gives rise to new and interesting physical and chemical properties, such as fluorescence enhancement and varied magnetic response. In addition, anisotropic nanomaterials aligned throughout the composite crystal have polarization dependent optical properties.

Simplex algorithms have been used to optimize for size, number and morphology of lysozyme and apoferritin crystals. This approach requires fewer experiments than the single-factor-at-a-time method or factorial designs and will be useful in conserving materials on the International Space Station. The simplex method has the possible advantage that it conserves on materials by reducing the number of experiments required to optimize a crystallization system. The process is iterative and exploratory and should allow optimum microgravity conditions to be determined which might very well be different from the optimum conditions on Earth. Because the simplex method uses simple mathematical operations to calculate the next set of crystallization conditions it will be easier for crystal growers to implement than factorial designs. Factorial experiments are based on varying all factors simultaneously at a limited number of factor levels. This results in a model that is used to determine the influence of each factor and their interactions. Factorial design experiments are especially useful at the beginning of an experimental study and as a screening tool to investigate a large number of factors. The simplex method is an optimization method which is model-independent and requires no fitting of models to data. Also, when applied to protein crystal growth the simplex method does not rely on an absolute quality score. Instead, with each iteration a comparison is made to the last experiment and the results are assigned as being "better or worse". In this study, commercially obtained apoferritin was purified from 65% monomeric apoferritin to 92% monomeric apoferritin by size exclusion chromatography. Simplex optimization found the best apoferritin crystals were obtained at 15 mg/ml apoferritin, 2.0% CdSO4, 25°C using the hanging drop vapor diffusion method of crystallization and at 24 mg/ml apoferritin, 1.5% CdSO4, 25°C using the containerless crystallization method. For lysozyme, the simplex method found the best crystals at 19 mg/ml lysozyme, 7.0% (w/v) NaCl, pH 4.0, 25°C using the hanging drop vapor diffusion method of crystallization. For both proteins, the optimum conditions were found with less than ten experiments using very little protein. Finally, we report that the factors to be considered in the successful application of this method to crystallization are the number of variables to be studied, the initial conditions, step size and analysis of crystal quality.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19182号 / 工博第4059号 / 新制||工||1626(附属図書館) / 32174 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 杉野目 道紀, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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