BIOMOLECULAR FOLDING/RECOGNITION PROBED BY KINETIC EPR

通过动力学 EPR 探测生物分子折叠/识别

• 批准号: 6722693
• 负责人: Charles P. Scholes
• 金额: $ 19.74万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6722693
• 关键词: CD4 molecule; RNA; conformation; cytochrome c; data collection methodology /evaluation; dielectric property; electron spin resonance spectroscopy; heat shock proteins; human immunodeficiency virus 1; lysozyme; molecular chaperones; molecular shape; nanotechnology; protein binding; protein folding; protein sequence; protein structure; stop flow technique; technology /technique development; time resolved data

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一种独一无二的高灵敏度流量和停止流量EPR来探测实时折叠和识别自旋标记的生物分子的识别，并从50微分钟内扩展到第二秒。要调查的领域是： ISO-1-CytoChrome c。这项工作建立在我们的高收益表达系统的基础上，用于外部位于ISO-1-环形c的外部，非扰动，半胱氨酸指导的突变体。使用单个突变体，我们将测量快速，亚数折叠的位置，时间尺度和激活能量。使用双标记的突变体和快速混合流EPR，我们将测量遥远的三级结构和螺旋的时间发展。 RNA蛋白折叠/识别。我们将研究茎环RNA-HIV-1核素蛋白复合物的折叠和识别。使用特定的逆转录病毒茎环RNA自旋在其5'末端标记并标记为HIV NCP7蛋白的自旋标记，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，一种分子伴侣。我们将通过热休克蛋白DNAK探测自旋标记的疏水肽，并适应该肽结合，以响应ATP诱导的DNAK的构象变化。 T4溶菌酶。使用Rapid-Mix亚数流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，并找到依次分开的残基的时间尺度，以达到附近的折叠构象。 技术发展。我们将改善基于介电谐振器的技术，以实现流量和田间扫描的最佳协调，最少的试剂使用甚至较短的死亡时间。