Proteins in the Molecular Mechanisms of Tear Film Formation

泪膜形成分子机制中的蛋白质

• 批准号: 8324530
• 负责人: BEN J GLASGOW
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8324530
• 关键词: Amino Acids; Binding; Binding Proteins; Circular Dichroism; Clinical; Computer software; Crystallography; Disease; Drug Delivery Systems; Engineering; Environment; Eye; Eye diseases; Film; Fluorescence; Goals; Grant; Health; Human; Laboratories; Learning; Ligand Binding; Ligands; Link; Lipid Binding; Maps; Medical; Methods; Modeling; Molecular; Molecular Conformation; Movement; Nuclear Magnetic Resonance; Outcome; Peptides; Pharmaceutical Preparations; Plant Roots; Population; Protein Engineering; Protein Region; Proteins; Public Health; Resolution; Role; Science; Side; Site; Solutions; Structure; Technology; Testing; Time; Tryptophan; Variant; Vertebral column; Work; base; cold temperature; conformer; design; eye dryness; flexibility; innovation; lipocalin 1; mutant; new technology; novel; protein function; restraint; success; synthetic peptide; tear proteins; tool

ABSTRACT The overall goal of this proposal is to learn the role of proteins in the molecular mechanisms in human tear film formation. The project has potential to significantly impact the way proteins are studied in solution. We propose to capitalize on the technologic innovations developed in our laboratory of site directed tryptophan fluorescence and circular dichroism to further this effort. In addition the project may provide the basis for protein engineering of tear lipocalin for specific eye treatments. Since tear lipocalin is decreased in dry eye, the study of its functions and mechanisms will provide a better understanding of how a major protein component of human tears functions normally and what is needed to treat dry eye disease. The proposal has 2 specific aims: 1: Elucidation of tryptophan rotamer configurations in tear lipocalin by site directed tryptophan fluorescence lifetimes. 2. To deconvolve the near UV Circular Dichroism (CD) tryptophan spectra to identify specific loop conformers in tear lipocalin. AIM1. While amino acid rotamers are fundamental to mechanisms of protein function, they can only be deduced indirectly by nuclear magnetic resonance (NMR) and only for some proteins. Crystallography usually can assign one rotamer (the most populated one) for each amino acid residue and is limited to only the very few proteins which have exceedingly high resolution crystal structures. This proposal aims to create a direct method to observe rotamers of tryptophan in proteins in solution. While similar approaches were started in small synthetic peptides, success is lacking in complete natural proteins. We propose to use the backbone restraints of tear lipocalin combined our expertise in time resolved site directed tryptophan fluorescence to resolve rotamer populations in lifetime fluorescence. Success will provide a needed way to probe backbone and amino acid side chain constraints of rotamers in all proteins. AIM 2. We will test the hypothesis that low temperature UV CD tryptophan spectra will resolve conformational movement of the AB loop of tear lipocalin. The flexible loop regions in proteins, such as the loop AB of tear lipocalin, are critical to ligand binding but can not be resolved in most crystal structures. NMR is limited to indirect information and only in some proteins. By combining low temperature near UV site directed tryptophan CD and novel multi-variant spectral deconvolution software the vibronic structure of the loop can be for the first time educed. Generated will be an important tool for identifying loop conformations that are critical to ligand binding to aid our understanding of the critical loop function of tear lipocalin and many other proteins. The information is critical to engineering proteins to treat general medical as well as eye diseases.

抽象的 该提议的总体目标是学习蛋白质在人泪膜中分子机制中的作用 形成。该项目有可能显着影响溶液中蛋白质的研究方式。我们建议 利用我们的网站实验室指导的色氨酸实验室开发的技术创新 荧光和圆二色性，以进一步努力。此外，该项目可能为 泪液脂肪蛋白的蛋白质工程用于特定眼科处理。由于干眼症中撕裂脂蛋白降低了 研究其功能和机制将更好地理解如何的主要蛋白质成分 人眼泪正常起作用，以及治疗干眼症所需的东西。该提案有2个具体目标： 1：阐明色氨酸旋转型在撕裂脂肪蛋白中的构型，由特色的色氨酸阐明 荧光寿命。 2。对近距离紫外圆二色性（CD）色氨酸光谱进行解析至 确定泪液中的特定环构象体。 AIM1。尽管氨基酸旋转剂是蛋白质功能机制的基础，但它们只能是 通过核磁共振（NMR）间接推导，仅针对某些蛋白质推导。晶体学通常 可以为每个氨基酸残留物分配一个旋转A（人口最多），仅限于 很少有具有极高分辨率晶体结构的蛋白质。该建议旨在创建一个直接的 观察溶液中蛋白质中色氨酸旋转剂的方法。虽然开始使用类似的方法 小型合成肽，完全自然蛋白缺乏成功。我们建议使用骨干 撕裂脂蛋白的限制结合了我们在时间分辨率的专业知识，将色氨酸荧光定向到 在寿命荧光中解析旋转群体。成功将为探测骨干的必要方法 和所有蛋白质中旋转剂的氨基酸侧链约束。 AIM 2。我们将测试低温紫外CD色氨酸光谱将解决构象的假设 泪液lipocalin的AB环的运动。蛋白质中的柔性环区域，例如撕裂的环路AB Lipocalin，对配体结合至关重要，但在大多数晶体结构中无法解决。 NMR仅限于 间接信息，仅在某些蛋白质中。通过将紫外线附近的低温结合到定向色氨酸 CD和新型的多变体光谱反卷积软件可为第一个循环的振动结构 教育时间。生成将是识别对配体至关重要的循环构象的重要工具 结合以帮助我们理解泪液lipocalin和许多其他蛋白质的关键环路功能。这 信息对于工程蛋白至关重要，以治疗一般医疗和眼部疾病。