BIOMOLECULAR FOLDING/RECOGNITION PROBED BY KINETIC EPR

通过动力学 EPR 探测生物分子折叠/识别

基本信息

批准号：
7925180
负责人：
Charles P. Scholes
金额：
$ 7.12万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-01-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：这项工作的目的是利用我们独一无二的高灵敏度流动和停流 EPR 来探测自旋标记生物分子的实时折叠和识别，其时间分辨率从50微秒到秒。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表达系统的基础上，用于 iso-1-细胞色素 c 的外部定位、非扰动、半胱氨酸定向突变体。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。通过双标记突变体和快速混合流 EPR，我们将测量远处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' 末端进行自旋标记的特定逆转录病毒茎环 RNA 和自旋标记的 HIV NCP7 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。我们将探讨热休克蛋白 DnaK 对自旋标记疏水性肽的识别，以及该肽结合对 ATP 诱导的 DnaK 构象变化的适应性。 T4 溶菌酶。使用快速混合亚毫秒流 EPR，我们将研究双标记 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。我们将改进基于介电谐振器的技术，以实现流场和场扫描的最佳协调、最少的试剂使用以及更短的死区时间。