THE REFOLDING OF THE DINUCLEOTIDE FOLD

观察变性蛋白复性动力学的方法学 蛋白质的天然构象将被开发出来。年的下降 有效水动力体积将使用排除来观察。 高压层析。柱子的长度，弹性 ，并且色谱组件的几何形状将是 优化以便于通常测量动力学参数 在低温下观察到小蛋白的折叠。一个 将对仪器中的停流圆二向色性进行测试和改进 观察次生结构元素的形成及其作用 集合。一种快速扫描停流吸收和荧光的方法 光谱仪也将进行测试，以观察环境的变化 折叠过程中的内在和外在生色团。 含蛋白质折叠机理的研究 二核苷酸折叠、硫氧还蛋白及其变体将继续存在。 我们将详细研究稳定的折叠中间体的结构。 丁二烯顺反异构化反应的平衡和动力学参数 将测量Proline 76。额外的中间表格将是 通过多路混合协议进行搜索。它的稳定性和折叠性 将研究单个球状域以及它们的作用 折叠机构中的关联性。这种蛋白质的变种将是 由位点特异性突变构建，以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.