TIME-RESOLVED FLUORESCENCE STUDIES OF PROTEIN FOLDING

双动力学荧光停流光谱法将被结合 通过光谱靶向定点诱变和化学 操纵，以便实验性地检查蛋白质折叠途径。 荧光实验是在能够 同时实时检测五个独立的数据轴， 蛋白质折叠反应（皮秒/纳秒，毫秒/秒， 平行强度，垂直强度，多重发射 波长）。 高信噪比时间分辨各向异性， 利用每毫秒获得的荧光寿命， 直接测量中间折叠态的基本性质 proteins. 实验工作集中在检查 酵母磷酸甘油酸激酶（PGK）和E.大肠杆菌α- 色氨酸合酶亚基（α-TS.） 以下问题 （1）什么是时间尺度？时间尺度是什么？ 展开状态下爱因斯坦粒子的皮秒/纳秒局域运动 变成“耦合”到原生状态的全局旋转 折叠反应2)蛋白质中的所有结构域（或子域） 限制色氨酸运动的动力学一致吗3)是什么 初始塌陷蛋白质状态的流体动力学半径，以及 人口“过度膨胀”的国家？4)你带了什么- “这个最初坍塌的结构？5)时间尺度是什么 在PGK中，类似于本地人的距离的展开在： α-螺旋？，相邻的α-螺旋？一个单一的极端 域？穿过两个域之间的铰链同时使用多站点和单个 色氨酸停流各向异性和多点停流能- 传输测量，毫秒级的”结构和动态”运动- 将确定蛋白质折叠的图片。 实时测量 旋转动力学、分子内距离和溶剂 强调可访问性。 一种新型“双动能”的设计 介绍了一种脉冲幅度分析模数转换器， 将并行ADC与多维可索引直方图相结合 存储器阵列，以增加双- 动力学方法通过另一个10 X的因子。

Double-kinetic fluorescence stopped-flow spectroscopy will be combined with spectroscopy-targeted site directed mutagenesis and chemical manipulation in order to experimentally examine protein folding pathways. Fluorescence experiments are performed on an instrument capable of simultaneous detection of five independent data axes in real-time during protein folding reactions (picosecond/nanosecond, millisecond/second, parallel intensity, perpendicular intensity, multiple emission wavelengths). High signal-to-noise time-resolved anisotropies and fluorescence lifetimes obtained every millisecond are utilized to directly measure fundamental properties of intermediate folded states of proteins. Experimental efforts are concentrated on the examination of the folding of yeast phosphoglycerate kinase (PGK) and E. coli alpha- subunit of tryptophan synthase (alpha-TS.) The following questions are being directly examined: 1) What is the exact time-scale upon which the picosecond/nanosecond local motions of tryptophans in the unfolded state become "coupled" to the global rotation of the native state during a folding reaction. 2) Do all domains (or subdomains) within a protein restrict this tryptophan motion with identical kinetics? 3) What is the hydrodynamic radius of initially collapsed protein states, and transiently populated "overexpanded" states? 4) Do tryptophans "pack- into" this initially collapsed structure? 5) What is the time-scale for the unfolding of native-like distances in PGK between: both ends of an alpha-helix?, adjacent alpha-helices?, the extreme ends of a single domain?, across a hinge between two domains? Using both multi-site single tryptophan stopped-flow anisotropy and multi-site stopped-flow energy- transfer measurements, millisecond" structural-and-dynamic" motion- pictures of protein folding will be determined. Real-time measurements of rotational dynamics, intramolecular distances, and solvent accessibility are emphasized. The design of a new "double-kinetic" pulse-height-analyisis analog-to-digital-converter is described, which combines parallel ADC's with multi-dimensional indexable histogramming memory arrays, to increase the inherent timing resolution of the double- kinetic method by another factor of 10X.