Proteins in the Molecular Mechanisms of Tear Film Formation

描述(由申请人提供)：这项建议的总体目标是了解蛋白质在人类泪膜形成的分子机制中的作用。该项目有可能对蛋白质在溶液中的研究方式产生重大影响。我们建议利用我们实验室开发的定点定向色氨酸荧光和圆二色谱的技术创新来进一步推动这一努力。此外，该项目还可能为泪液脂质蛋白用于特定眼部治疗的蛋白质工程提供基础。由于干眼症患者泪液中的脂钙蛋白减少，对其功能和机制的研究将有助于更好地了解人类泪液的主要蛋白质成分是如何正常发挥作用的，以及治疗干眼症需要什么。该提案有两个具体目标：1：通过位点定向的色氨酸荧光寿命来阐明泪液Lipocalin中色氨酸旋转异构体的配置。2.对近紫外圆二色谱(CD)色氨酸光谱进行去卷积，以确定泪液Lipocalin中特定的环状构象。AIM1.虽然氨基酸旋转体是蛋白质功能机制的基础，但它们只能通过核磁共振(NMR)间接推断，而且只能对某些蛋白质进行推断。结晶学通常可以为每个氨基酸残基指定一个旋转异构体(最多的一个)，并且仅限于极少数具有极高分辨率晶体结构的蛋白质。这项提议旨在创造一种直接的方法来观察溶液中蛋白质中色氨酸的旋转异构体。虽然类似的方法已经在小的合成多肽中开始，但在完整的天然蛋白质中缺乏成功。我们建议使用泪液Lipocalin的骨架限制结合我们在时间分辨定点定向色氨酸荧光方面的专业知识来解析终身荧光中的旋转体群。该研究的成功将为探索所有蛋白质中旋转异构体的骨架和氨基酸侧链限制提供必要的途径。目的2.我们将验证一种假设，即低温UV CD色氨酸光谱将分解泪液Lipocalin的AB环的构象运动。蛋白质中的柔性环区域，如tear Lipocalin的环AB，是配体结合的关键，但在大多数晶体结构中不能被分辨。核磁共振仅限于间接信息，而且仅限于某些蛋白质。将近紫外区低温定向色氨酸CD与新型多变量光谱去卷积软件相结合，首次推导出该环的振动结构。产生将是一个重要的工具，以确定环构象是关键的配体结合，以帮助我们了解关键的环功能的泪液脂结合蛋白和许多其他蛋白质。这些信息对于改造蛋白质来治疗普通内科和眼科疾病至关重要。 公共卫生相关性：这个项目的相关性是了解人类眼泪中蛋白质的复杂分子相互作用。其目标是开发新的技术来询问泪水中的分子机制，同时了解泪液蛋白如何在健康和疾病中发挥作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The relevance of this project is to learn the intricate molecular interactions of proteins in human tears. The goal is to develop new technologies to interrogate the molecular mechanisms in tears and at the same time learn how tear proteins function in health and disease.