FLUORESCENCE STUDIES OF PEPTIDE STRUCTURE AND DYNAMICS

小而灵活的多肽介导了广泛的生物过程 从大脑到肠道的人体组织中。即使是一个单独的多肽也能唤起 根据目标组织的不同，反应也不同。在许多可能的情况下 溶液中的构象，只有几个具有生物活性 由特定受体识别的构象。的结构确定 因此，柔性多肽是结构类药物的一个重要挑战 设计。这项提议发展了荧光技术，用于研究 柔性多肽在溶液和膜中的结构和动力学 受体复合体。我们的方法的特点是设计了新的荧光 模拟自然产生的芳香族氨基酸的探测器 对多肽结构和活性的干扰最小。这些探头依次 是理解荧光之间关系的门户 寿命和基态结构。激发态的性质是 由时间相关的单光子计数确定。基态 结构由X-射线结晶学、核磁共振和分子测定确定 机械师。 色氨酸光物理的基础研究将剖析这个复合体 吲哚发色团的光物理学。负责的非辐射过程 对于荧光寿命的环境敏感性，包括： 溶剂猝灭与激发态质子和电子转移 反应。用于识别、分离和量化多个 竞争性的非辐射过程将被用来绘制所有可能的 吲哚环与氨基酸官能团的相互作用猝灭 组。荧光猝灭机制将由溶剂来描述。 同位素效应、温度依赖性和光化学同位素交换 实验。第二代受限色氨酸衍生物 模拟多肽中所有重要的色氨酸构象将是 合成的。这些探头将定义接近度和方向 多肽对色氨酸荧光猝灭的要求及其机制 债券。 分子内猝灭的距离和取向要求 色氨酸的荧光由氨基酸官能团组成 对真实的多肽进行了系统的研究。约束和本地化 侧链猝灭吲哚的色氨酸衍生物和氨基酸 荧光将被结合到刚性生长抑素类似物中。 我们将对猝灭机制进行描述。激发态寿命将 与高分辨率核磁共振确定的溶液构象有关 自由能分子力学。苯环化色氨酸衍生物 将被开发用于探测复杂环境中的多肽结构，如 作为膜受体。具有丙烯酰胺类官能团的氨基酸 将被合并为苯环吲哚的分子内猝灭剂 荧光。这项工作测试了多肽结构的方法学 已知结构的多肽中的测定。未来的工作将涉及 柔性多肽在溶液和膜结合中的应用 感受器。

Small, flexible peptides mediate a broad spectrum of biological processes in human tissues from brain to gut. Even a single peptide can evoke different responses depending on the target tissue. Of the many possible conformations in solution, only a few will be biologically active conformers recognized by specific receptors. Structural determination of flexible peptides is thus an important challenge for structure-based drug design. This proposal develops fluorescence techniques for studying the structure and dynamics of flexible peptides in solution and in membrane receptor complexes. Our approach features the design of novel fluorescence probes that mimic the naturally occurring aromatic amino acids while minimally perturbing peptide structure and activity. These probes in turn are the gateway to understanding the relationship between fluorescence lifetime and ground-state structure. Excited-state properties are determined by time-correlated single photon counting. Ground-state structure is determined by X-ray crystallography, NMR, and molecular mechanics. Fundamental studies of tryptophan photophysics will dissect the complex photophysics of the indole chromophore. Nonradiative processes responsible for the environmental sensitivity of the fluorescence lifetime include: solvent quenching and excited-state proton and electron transfer reactions. Strategies for identifying, separating, and quantifying several competitive nonradiative processes will be used to map all possible quenching interactions between the indole ring and amino acid functional groups. Fluorescence quenching mechanisms will be delineated by solvent isotope effect, temperature dependence, and photochemical isotope exchange experiments. Second generation constrained tryptophan derivatives that mimic all significant tryptophan conformations in peptides will be synthesized. These probes will define the proximity and orientation requirements and mechanism of tryptophan fluorescence quenching by peptide bonds. Distance and orientation requirements for intramolecular quenching of tryptophan fluorescence by amino acid functional groups will be systematically studied in real peptides. Constrained and benzannulated tryptophan derivatives and amino acids whose side chains quench indole fluorescence will be incorporated into rigid somatostatin analogs. Quenching mechanisms will be characterized. Excited-state lifetimes will be related to solution conformations determined by high resolution NMR and free energy molecular mechanics. A benzannulated tryptophan derivative will be developed to probe peptide structure in complex environments, such as membrane receptors. Amino acids with acrylamide-like functional groups will be incorporated as intramolecular quenchers of benzannulated indole fluorescence. This work tests the methodology for peptide structure determination in peptides of known structure. Future work will involve application to flexible peptides in solution and bound to membrane receptors.