Proteins in the Molecular Mechanisms of Tear Film Formation

描述（由申请人提供）：本提案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。该项目有可能显著影响蛋白质在溶液中的研究方式。我们建议利用我们实验室开发的技术创新的现场定向色氨酸荧光和圆二色性，以进一步这方面的努力。此外，该项目可能为特定眼部治疗的泪液脂质运载蛋白的蛋白质工程提供基础。由于泪液脂质运载蛋白在干眼症中减少，因此对其功能和机制的研究将更好地了解人类泪液的主要蛋白质组分如何正常发挥作用以及治疗干眼症所需的内容。该提案有2个具体目标：1：通过位点定向色氨酸荧光寿命阐明泪液脂质运载蛋白中色氨酸旋转异构体构型。2.对近紫外圆二色性（CD）色氨酸光谱进行去卷积，以鉴定泪液脂质运载蛋白中的特定环构象。AIM 1.虽然氨基酸旋转异构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，并且仅适用于某些蛋白质。晶体学通常可以为每个氨基酸残基分配一个旋转异构体（最常见的一个），并且仅限于极少数具有极高分辨率晶体结构的蛋白质。该建议旨在建立一种直接观察溶液中蛋白质中色氨酸旋转异构体的方法。虽然类似的方法开始于小的合成肽，但在完整的天然蛋白质中缺乏成功。我们建议使用泪液脂质运载蛋白的骨架限制，结合我们在时间分辨位点定向色氨酸荧光方面的专业知识，来分辨终生荧光中的旋转异构体群体。成功将提供一个必要的方式来探测所有蛋白质中旋转异构体的骨架和氨基酸侧链限制。AIM 2.我们将测试低温UV CD色氨酸光谱将解析泪液脂质运载蛋白的AB环的构象运动的假设。蛋白质中的柔性环区域，例如泪液脂质运载蛋白的环AB，对配体结合至关重要，但在大多数晶体结构中无法解析。NMR仅限于间接信息，并且仅在某些蛋白质中。结合低温近紫外定位色氨酸圆二色谱和新型多变量光谱解卷积软件，首次导出了环的电子振动结构。产生的将是一个重要的工具，用于识别环构象，是至关重要的配体结合，以帮助我们了解的关键环功能的眼泪脂质运载蛋白和许多其他蛋白质。这些信息对于工程蛋白质治疗一般医学和眼科疾病至关重要。 公共卫生相关性：该项目的相关性是了解人类泪液中蛋白质的复杂分子相互作用。目标是开发新技术来询问眼泪中的分子机制，同时了解眼泪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.