BIOMOLECULAR FOLDING/RECOGNITION PROBED BY KINETIC EPR

描述（由申请人提供）：这项工作的目的是利用我们的一种，高灵敏度流动和停流EPR探测自旋标记的生物分子的真实的时间折叠和识别，时间分辨率从50微秒延伸到秒。需要调查的领域是： Iso-1-细胞色素c.这项工作建立在我们的高产量表达系统的外部定位，非干扰，半胱氨酸定向突变体的异-1-细胞色素c。对于单个突变体，我们将测量快速亚毫秒折叠的位置、时间尺度和活化能。利用双标记突变体和快速混合流EPR，我们将测量远端三级结构和螺旋的时间发展。 RNA-蛋白质折叠/识别。我们将研究茎环RNA-HIV-1核衣壳蛋白复合物的折叠和识别。使用在其5'末端自旋标记的特定逆转录病毒茎环RNA和自旋标记的HIV NCP 7蛋白，我们将测量导致最终RNA-蛋白质复合物的结构变化的时间过程。 DnaK分子伴侣我们将探测热休克蛋白DnaK的自旋标记的疏水肽的识别和这种肽结合的适应响应于ATP诱导的DnaK的构象变化。 T4溶菌酶。使用快速混合亚毫秒流EPR，我们将研究双标记的T4溶菌酶，以找到蛋白质螺旋形成的时间尺度，并找到序列中相距很远的残基达到其附近的折叠构象的时间尺度。 技术开发。我们将改进基于介电谐振器的技术，以实现流量和场扫描的最佳协调，最大限度地减少试剂的使用，甚至缩短死区时间。

DESCRIPTION (provided by applicant): The purpose of this work is to exploit our one-of-a-kind, high sensitivity flow and stopped-flow EPR to probe the real time folding and recognition of spin labeled biomolecules with a time resolution extending from 50 microseconds to seconds. The areas to be investigated are: Iso-1-Cytochrome c. This work builds on our high yield expression system for externally located, nonperturbing, cysteine-directed mutants of iso-1-cytochrome c. With single mutants, we will measure the location, time scale, and activation energy of rapid, submillisecond folding. With bi-labeled mutants and rapid-mix flow EPR we will measure the time development of distant tertiary structure and helices. RNA-Protein Folding/Recognition. We will study folding and recognition of a stem-loop RNA-HIV-1 nucleocapsid protein complex. Using a specific retroviral stem loop RNA spin labeled at its 5' terminal and spin labeled HIV NCP7 protein, we will measure the time course for structural changes leading to the final RNA-protein complex. DnaK, a Molecular Chaperone. We will probe recognition of a spin-labeled hydrophobic peptide by the heat shock protein DnaK and adaptation of this peptide binding in response to ATP-induced conformational change of DnaK. T4 Lysozyme. Using rapid-mix submillisecond flow EPR, we will study bi-labeled T4 lysozyme to find the time scale for protein helix formation and to find the time scale for residues far apart in sequence to reach their nearby folded conformation. Technical Development. We will improve our dielectric resonator based technology to achieve optimum coordination of flow and field sweep, minimal use of reagents, and even shorter dead times.