Proteins in the Molecular Mechanisms of Tear Film Formation

摘要 这项建议的总体目标是了解蛋白质在人类泪膜分子机制中的作用。 队形。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 利用我们的现场定向色氨酸实验室开发的技术创新 荧光和圆二色谱进一步推动了这一努力。此外，该项目还可以为 泪液Lipocalin的蛋白质工程用于特定的眼科治疗。由于干眼症患者的泪液脂结合蛋白减少， 对其功能和机制的研究将有助于更好地理解细胞内的一种主要蛋白质成分是如何 人类的泪水功能正常，治疗干眼症需要什么。该提案有两个具体目标： 1：用定点色氨酸研究泪液Lipocalin中色氨酸旋转异构体的构型 荧光寿命。2.将近紫外圆二色(CD)色氨酸光谱去卷积为 鉴定眼泪脂钙蛋白中的特定环状构象。 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 由核磁共振间接推断，仅对某些蛋白质有效。结晶学通常 可以为每个氨基酸残基分配一个旋转异构体(最多的一个)，并且仅限于 很少有具有极高分辨率晶体结构的蛋白质。这项提议旨在创造一个直接的 方法观察溶液中蛋白质中色氨酸的旋转异构体。虽然类似的方法在 小的合成肽，成功是缺乏完整的天然蛋白质。我们建议使用主干 泪液Lipocalin的抑制结合了我们在时间分辨部位定向色氨酸荧光的专业知识 在终身荧光中解析旋转分子种群。成功将提供探测骨干网所需的方式 以及所有蛋白质中旋转异构体的氨基酸侧链限制。 目的2.我们将验证低温UV CD色氨酸光谱将解析构象的假设 泪液脂钙蛋白AB环的运动。蛋白质中的柔性环区域，如撕裂的环AB Lipocalin是配体结合的关键，但在大多数晶体结构中不能被分解。核磁共振仅限于 间接信息，并且仅存在于某些蛋白质中。通过结合紫外光附近的低温定向色氨酸 CD和新颖的多变量光谱反褶积软件，可首次实现环路的振动结构 时间到了。生成的将是识别对配体至关重要的环构象的重要工具 结合以帮助我们理解泪液Lipocalin和许多其他蛋白质的关键环功能。这个 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。

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.