TIME-RESOLVED FLUORESCENCE STUDIES OF PROTEIN FOLDING

双动力学荧光停流光谱分析将结合 利用光谱靶向定点突变和化学 操控以实验地检查蛋白质折叠途径。 荧光实验是在一台能够 实时同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和 利用每毫秒获得的荧光寿命来 直接测量中间折叠态的基本性质 蛋白质。实验的努力集中在考试上 酵母磷酸甘油酸激酶(PGK)与大肠杆菌α-DNA的折叠 色氨酸合成酶亚基(α-TS.)以下是以下问题 被直接检查：1)准确的时间尺度是什么 色氨酸在展开状态下的皮秒/纳秒局部运动 变得与本机状态的全局旋转在 折叠反应。2)蛋白质中的所有结构域(或亚结构域) 用相同的动力学限制色氨酸运动？3)什么是 最初塌陷的蛋白质状态的流体动力学半径，以及 短暂存在的“过度膨胀”状态？4)色氨酸会不会“打包”？ 这个最初倒塌的结构？5)时间尺度是什么？ 在PGK中展开的原生距离： α-螺旋？，相邻的α-螺旋？，单个 域？，跨越两个域之间的铰链？同时使用多站点和单站点 色氨酸停流各向异性和多点停流能量- 传递测量，毫秒“结构和动力”运动- 蛋白质折叠的图片将被确定。实时测量 旋转动力学、分子内距离和溶剂 强调了可及性。一种新型“双动”机构的设计 介绍了一种脉冲幅度分析型模数转换器，它 将并行ADC与多维可索引组织图相结合 存储器阵列，以提高双倍时钟的固有时序分辨率 动力学方法的另一个因子是10倍。

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.