Spectroscopic markers for blue-fluorescing tryptophan in proteins

描述（由申请人提供）：对于导致蛋白质中色氨酸荧光发射猝灭和蓝移的因素的理解存在根本性差距。长期目标是定义一组光谱标记，以便任何色氨酸环境和结构都可以轻松破译和量化。本提案的目的是了解不同的 pH 因素（就短色氨酸肽而言）以及疏水性和空间位阻（就蛋白质色氨酸而言）如何导致荧光发射光谱蓝移且发射强度降低。该应用的中心假设是，色氨酸的紫外共振拉曼振动带可用于破译导致蛋白质色氨酸和色氨酸肽荧光发射光谱中出现非典型蓝色荧光特征的潜在结构因素。紫外共振拉曼谱带比荧光发射谱带更清晰、分辨率更好，并且许多谱带已经与特定的色氨酸特征（例如吲哚胺上的氢键）相关联。这项研究的基本原理是，一旦使色氨酸蓝色荧光猝灭的潜在结构特征和环境变得明确，荧光发射光谱法的蛋白质表征将产生更有用和更明确的信息：所谓的结构标记。在强有力的初步数据的背景下，我们计划测试我们的中心假设，并通过追求以下两个具体目标来实现本应用的目标：1）确定蓝色荧光色氨酸肽和蛋白质共有的结构基序； 2) 解决 1Bb、1La 和 1Lb 过渡偶极矩对蓝色荧光色氨酸肽和蛋白质吸收带的重叠影响。第一个目标是通过紫外共振拉曼光谱研究一组蓝色荧光肽、一组互补的红移肽和蓝色荧光蛋白。在已知的紫外共振拉曼能带结构关联的指导下，通过分析预计会出现导致荧光蓝移的因素模式。第二个目标是对含色氨酸的肽进行荧光激发各向异性测量，以解析 1Bb、1La 和 1Lb 跃迁偶极矩对 220 nm 和 280 nm 吸收带的贡献。该提议具有创新性，因为紫外共振拉曼结果尚未系统地应用于蓝色荧光蛋白的研究。此外，高能 1Bb 跃迁偶极矩的能量分布和位置此前尚未包含在 1La 和 1Lb 吸收带分布的研究中。拟议的研究意义重大，因为将提高人们对色氨酸蛋白荧光发射光谱特征的理解。因此，这项研究将对未来正常和突变蛋白质结构的研究产生积极影响，从而影响药物设计以对抗异常蛋白质引起的疾病。 公共卫生相关性：突变和错误折叠的蛋白质是人类疾病的致病因素。为了设计治疗蛋白质相关疾病的药物，必须首先了解异常蛋白质的结构和功能。本研究旨在通过开发荧光发射光谱技术来增进对疾病相关蛋白质的理解，该技术可探测天然存在的荧光氨基酸色氨酸的结构和环境。

DESCRIPTION (provided by applicant): There is fundamental gap in the understanding of the factors causing quenched and blue-shifted fluorescence emission of tryptophan in proteins. The long term goal is to define a set of spectroscopic markers such that any tryptophan environment and structure can be readily deciphered and quantified. The objective of this proposal is to understand how the disparate factors of pH, in the case of short tryptophan peptides, and hydrophobicity and steric hindrance, in the case of proteinaceous tryptophans, both result in blue-shifted fluorescence emission spectra with reduced emission intensity. The central hypothesis of this application is that UV resonance Raman vibrational bands for tryptophan can be used to decipher the underlying structural factors that cause the atypical blue fluorescence features seen in both proteinaceous tryptophan and tryptophan peptide fluorescence emission spectra. UV resonance Raman bands are sharper, and better-resolved than fluorescence emission bands, and many have already been associated with specific tryptophan features such as hydrogen bonding at the indole amine. The rationale for the proposed research is that once the underlying structural features and environment responsible for the quenched, blue fluorescence of tryptophan is made explicit, protein characterization by fluorescence emission spectroscopy will yield more useful and explicit information: so-called structural markers. In the context of strong preliminary data, we plan to test our central hypothesis and accomplish the objectives of this application by pursuing the following two specific aims: 1) Determine the structural motifs common to blue-fluorescing tryptophan peptides and proteins; and 2) Resolve the overlap of 1Bb, 1La and 1Lb transition dipole moment contributions to the absorption bands of blue- fluorescing tryptophan peptides and proteins. Under the first aim, a set of blue-fluorescing peptides, a complementary set of red-shifted peptides and blue-fluorescing proteins will be studied by UV resonance Raman spectroscopy. Guided by known UV resonance Raman band-structure associations, a pattern of factors contributing to the blue-shift of fluorescence are expected from analysis. Under the second aim, fluorescence excitation anisotropy measurements on the tryptophan-containing peptides will be used to resolve the contribution of the 1Bb, 1La and 1Lb transition dipole moments to the 220 nm and 280 nm absorption bands. This proposal is innovative because UV resonance Raman results have not been applied systematically to the study of blue-fluorescing proteins. Also, the energy profile and position of the high energy 1Bb transition dipole moment has not previously been included in studies of the 1La and 1Lb absorption band profile. The proposed research is significant because a higher level of understanding of the features seen in the fluorescence emission spectra of tryptophan-bearing proteins will result. Therefore, this study will have a positive impact on future studies of normal and mutant protein structure, and therefore influence drug design to combat disease resulting from aberrant proteins. PUBLIC HEALTH RELEVANCE: Mutant and misfolded proteins are disease-causing agents in humans. In order to design drugs to treat protein-related diseases, the structure and function of the aberrant proteins must be first understood. This study aims to advance the understanding of disease-related proteins through development of the technique, fluorescence emission spectroscopy, which probes the structure and environment of the naturally-occurring fluorescent amino acid, tryptophan.