FLUORESCENCE STUDIES OF PEPTIDE STRUCTURE AND DYNAMICS

小而灵活的肽介导了广泛的生物过程 从大脑到肠道的人体组织中。即使是一个单一的肽可以唤起 不同的反应取决于目标组织。的许多可能 溶液中的构象，只有少数将具有生物活性 被特定受体识别的构象异构体。的结构测定 因此，柔性肽是基于结构的药物的重要挑战 设计这项建议发展了荧光技术，用于研究 柔性肽在溶液和膜中结构和动力学 受体复合物我们的方法的特点是设计新的荧光 模拟天然存在的芳香族氨基酸， 最低限度地干扰肽结构和活性。这些探测器依次 是理解荧光与 寿命和基态结构。 激发态性质是 通过时间相关单光子计数确定。基态 通过X射线晶体学、NMR和分子生物学确定结构。 力学 色氨酸生物物理学的基础研究将剖析复合物 吲哚发色团的电子物理学。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.识别、分离和量化几种方法的策略 竞争性的非辐射过程将被用来映射所有可能的 吲哚环和氨基酸官能团之间的猝灭相互作用 组荧光猝灭机制将通过溶剂来描述 同位素效应、温度依赖性和光化学同位素交换 实验第二代限制性色氨酸衍生物， 模拟肽中所有重要的色氨酸构象， 合成了这些探针将定义距离和方向 多肽对色氨酸荧光猝灭的要求及机理 债券 分子内猝灭的距离和取向要求 色氨酸荧光由氨基酸官能团将是 在真实的缩氨酸中进行了系统的研究。限制性和苯环化 色氨酸衍生物和侧链淬灭吲哚的氨基酸 荧光将掺入刚性生长抑素类似物中。 淬火机制的特点。激发态寿命将 与通过高分辨率NMR确定的溶液构象有关， 自由能分子力学一种苯环化色氨酸衍生物， 将开发用于在复杂环境中探测肽结构，例如 as membrane膜receptor受体.具有丙烯酰胺样官能团的氨基酸 将作为苯环化吲哚的分子内猝灭剂掺入 荧光。这项工作测试了肽结构的方法 已知结构的肽的测定。今后的工作将涉及 应用于溶液中的柔性肽和与膜结合的柔性肽 受体。

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.