AFFINITY CHROMATOGRAPHY FOR THE DINUCLEOTIDE FOLD

变性蛋白质复性动力学的观察方法 将蛋白质开发成它们的天然构象。 减少 有效流体动力学体积将使用排除法来观察 在高压下进行色谱分析。 柱子的长度， 的矩阵，和色谱组件的几何形状将是 被优化以便于动力学参数的测量 在低温下小蛋白质的折叠中观察到。 一 对仪器中的停流圆二色性进行了测试和改进 观察二级结构元件的形成及其 组装件. 快速扫描停流吸光度和荧光 光谱仪也将进行测试，以观察环境的变化， 内在和外在的生色团在折叠。 研究了含蛋白质的折叠机理， 二核苷酸折叠、硫氧还蛋白及其变体将继续。 一个稳定的折叠中间体的结构将被详细检查。 本文测定了顺反异构化反应的平衡参数和动力学参数 测量脯氨酸76。 其他中间形式将 通过多重混合协议搜索。 的稳定性和折叠性 将检查单个球状域以及它们的作用。 在折叠机构中的关联。 蛋白质的变体将 通过位点特异性突变来扰乱折叠的各个方面， 机制

Methodology for observation of the kinetics of the refolding of denatured proteins into their native conformation will be developed. The decrease in the effective hydrodynamic volume will be observed using exclusion chromatography at high pressure. The length of the column, the resiliance of the matrix, and the geometry of the chromatographic assembly will be optimized to facilitate measurement of the kinetic parameters typically observed in the folding of small proteins at low temperature. A stopped-flow circular dichroism in instrument will be tested and modified to observe the formation of secondary structural elements and their assembly. A rapid scan stopped-flow absorbance and fluorescence spectrometer will also be tested to observe changes in the environments of intrinsic and extrinsic chromophores during folding. Studies of the mechanism of the folding of a protein containing the dinucleotide fold, thioredoxin, and variants thereof will be continued. The structure of a stable folding intermediate will be examined in detail. The equilibrium and kinetic parameters of the cis/trans isomerization of proline 76 will be measured. Additional intermediate forms will be searched for by multimixing protocols. The stability and folding of the individual globular domains will be examined as well as the role of their association in the folding mechanism. Variants of the protein will be constructed by site specitic mutations to perturb aspects of the folding mechanism.