Spectroscopic markers for blue-fluorescing tryptophan in proteins

蛋白质中蓝色荧光色氨酸的光谱标记

• 批准号: 7848617
• 负责人: LAURA Jeanne JUSZCZAK
• 金额: $ 14.98万
• 依托单位: BROOKLYN COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7848617
• 关键词: Amines; Amino Acids; Anisotropy; Area; Behavior; Binding; C-terminal; Cities; Creutzfeldt-Jakob Syndrome; Data; Development; Dipeptides; Disease; Drug Design; Electronics; Ensure; Environment; Fluorescence; Fluorescence Spectroscopy; Funding; Future; Goals; Health; Human; Hydrogen Bonding; Hydrophobicity; Indoles; Knowledge; Link; Measurement; Medical; Mentors; Metals; Methods; Mission; Molecular; Molecular Conformation; Mutate; Mutation; N-terminal; New York; New York City; Outcome; Pattern; Peptides; Positioning Attribute; Principal Investigator; Prion Diseases; Prions; Process; Protein Engineering; Proteins; Publications; Publishing; Raman Spectrum Analysis; Reporter; Research; Research Personnel; Resources; Role; Seasons; Secure; Shapes; Solvents; Spectrum Analysis; Structure; Surface; Surgical Instruments; Techniques; Testing; Tryptophan; United States National Institutes of Health; Universities; Work; absorption; base; chromophore; college; combat; dipole moment; drug development; emission spectroscopy; fluorophore; innovation; interest; metropolitan; mutant; protein function; protein misfolding; protein structure; public health relevance; pyridine; structural biology; technique development; ultraviolet

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.

描述（由申请人提供）：在理解引起蛋白质中色氨酸荧光发射猝灭和蓝移的因素方面存在根本性差距。长期目标是定义一组光谱标记物，使得任何色氨酸环境和结构都可以容易地破译和定量。本提案的目的是了解pH的不同因素（在短色氨酸肽的情况下）以及疏水性和空间位阻（在蛋白质类色氨酸的情况下）如何导致发射强度降低的蓝移荧光发射光谱。本申请的中心假设是，色氨酸的UV共振拉曼振动带可用于破译导致在蛋白质色氨酸和色氨酸肽荧光发射光谱中看到的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，分辨率更高，许多谱带已经与特定的色氨酸特征（如吲哚胺上的氢键）相关。这项研究的基本原理是，一旦明确了色氨酸淬灭蓝色荧光的潜在结构特征和环境，通过荧光发射光谱法进行蛋白质表征将产生更有用和明确的信息：所谓的结构标记。在强有力的初步数据的背景下，我们计划通过追求以下两个特定目标来测试我们的中心假设并实现本申请的目标：1）确定发蓝色荧光的色氨酸肽和蛋白质的共同结构基序;以及2）解析1Bb、1 La和1 Lb跃迁偶极矩对发蓝色荧光的色氨酸肽和蛋白质的吸收带的贡献的重叠。在第一个目标下，一组蓝色荧光肽，一组互补的红移肽和蓝色荧光蛋白将通过紫外共振拉曼光谱进行研究。由已知的UV共振拉曼能带结构协会的指导下，从分析中预期有助于荧光蓝移的因素的模式。根据第二个目标，荧光激发各向异性测量的含色氨酸的肽将被用来解决的1Bb，1 La和1 Lb跃迁偶极矩的220 nm和280 nm的吸收带的贡献。这一建议是创新的，因为紫外共振拉曼的结果还没有被系统地应用到蓝色荧光蛋白质的研究。此外，能量分布和高能量1Bb跃迁偶极矩的位置以前没有包括在1 La和1 Lb的吸收带分布的研究。拟议的研究是有意义的，因为更高层次的了解的特点，看到在荧光发射光谱的蛋白质的携带的荧光。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的致病因子。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促进对疾病相关蛋白质的理解，该技术探测天然存在的荧光氨基酸色氨酸的结构和环境。