BIOMOLECULAR FOLDING/RECOGNITION PROBED BY KINETIC EPR

描述(申请人提供)：这项工作的目的是利用我们独一无二的、高灵敏度的流动和停止流动的EPR来探测自旋标记生物分子的实时折叠和识别，时间分辨率从50微秒延伸到几秒。将进行调查的领域包括： 这项工作建立在我们对iso-1-细胞色素c的外部定位、非扰动、半胱氨酸导向的突变体的高效表达系统的基础上。对于单个突变体，我们将测量快速、亚毫秒折叠的位置、时间尺度和激活能。利用双标记突变体和快速混合流EPR，我们将测量远距离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核衣壳蛋白复合体的折叠和识别。使用在其5‘端标记的特定逆转录病毒茎环RNA和标记的HIV NCP7蛋白，我们将测量导致最终RNA-蛋白质复合体的结构变化的时间过程。 DNAK，一个分子伴侣。我们将探索热休克蛋白DNAK对自旋标记的疏水性多肽的识别以及这种多肽结合对三磷酸腺苷诱导的DNAK构象变化的适应性。 T4溶菌酶。利用快速混合亚毫秒流EPR，我们将研究双标记T4溶菌酶，以寻找形成蛋白质螺旋的时间尺度，以及寻找相距较远的残基达到其附近折叠构象的时间尺度。 技术开发。我们将改进基于介质谐振器的技术，以实现流和场扫描的最佳协调，最大限度地减少试剂的使用，甚至更短的停机时间。

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.