MACROMOLECULAR DYNAMICS AND ASSEMBLY REACTIONS

大分子动力学和组装反应

• 批准号: 2572974
• 负责人: J HOFRICHTER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2572974
• 关键词: chemical kinetics; chemical models; conformation; fluorescence spectrometry; heme; lasers; macromolecule; molecular dynamics; peptides; photochemistry; photolysis; protein folding; protein structure; temperature jump; tryptophan

Time-resolved absorption and fluorescence spectroscopy are used to study the dynamics of protein structural changes subsequent to rapid mixing or excitation with short laser pulses. Molecular models for the protein dynamics are used to fit and interpret the measured data. A. We have succeed in implementing two new techniques which can be used to study the kinetics of a wide variety of processes, a rapid mixing technique capable of initiating reactions within 80 microseconds and a laser temperature jump experiment in which water is heated 10-20 K in less than 10 nanoseconds by an infrared laser pulse. B. The dynamics of short-lived states on the folding pathway of cytochrome c have been investigated using continuous-flow mixing and fluorescence quenching to monitor the tryptophan-heme distance. In the presence of imazole, which blocks binding of ligands to the sixth coordination site of the heme iron, mixing to low guanidine HCl produces a rapid collapse of the denatured state, occurring in less than 50 microseconds, followed by rapid exponential (< 1 millisecond) folding to the native conformation. Under conditions where non-native heme ligands can bind, rapid collapse is still observed, but the folding phase becomes at least biphasic as a result of 'trapping' of the denatured states by incorrect heme-ligand interactions. C. Laser temperature jump studies have been carried out on an 19- residue alanine peptide labeled at the n-terminus by methyl-amino benzoic acid. The fluorescence of this label appears to be highly dependent on hydrogen bonding of the carbonyl oxygen. We observe rapid melting of the helix with a maximum relaxation time of about 20 nanoseconds at the midpoint of the folding curve (35 C), while time- resolved infrared studies have found a relaxation time of about 100 nanoseconds under similar conditions. We attribute the faster relaxation to the fact that our probe monitors melting of the first turn of the helix, while the infrared measures the melting of the entire peptide.

时间分辨的吸收和荧光光谱用于 研究快速后的蛋白质结构变化的动力学 与短激光脉冲混合或激发。 分子模型 蛋白质动力学用于拟合和解释测量的数据。 答：我们成功地实施了两种新技术 用于研究各种过程的动力学，快速 混合技术能够在80范围内启动反应 微秒和激光温度跳跃实验，其中水为 红外激光脉冲在小于10纳秒的情况下加热10-20 K。 B.短暂状态在折叠途径上的动态 已经使用连续流混合和 荧光淬火以监测色氨酸血红素距离。 在 咪唑的存在，它阻断了配体与第六的结合 血红素铁的协调部位，混合到低鸟嘌呤HCl 产生变性状态的快速崩溃，发生在较少的情况下 超过50微秒，然后是快速指数（<1毫秒） 折叠到天然构象。 在非本地的条件下 血红素配体可以结合，仍然观察到快速崩溃，但是 折叠阶段至少由于“捕获”的结果而变得 变性状态通过不正确的血红素 - 配体相互作用。 C.激光温度跳跃研究是在19-上进行的 残留的丙氨酸肽由甲基氨基标记在N末端 苯甲酸。 该标签的荧光似乎很高 取决于羰基氧的氢键。 我们观察到 螺旋的快速熔化，最大放松时间约为20 在折叠曲线（35 C）的中点处的纳秒秒，而时间 - 解决的红外研究发现放松时间约为100 纳秒在类似条件下。 我们归因于更快的速度 放松的事实是，我们的探测器监测第一个的熔化 转弯螺旋，而红外线测量熔化 整个肽。