Serum Albumin: Binding and Transport of Pharmaceuticals and Functional Differences Derived from Divergent Evolution Characterized by Electron Paramagnetic Resonance (EPR) Spectroscopy

建立在最近为研究血清白蛋白而开发的基于EPR(电子顺磁共振)的研究平台的基础上，这项建议有两个相互关联的主要目标。第一个目标是了解血清白蛋白(人和动物，例如牛)在结合小的药物活性分子和与膜相互作用时的结构可塑性和动力学。这将使用EPR光谱学实现，并将有助于弥合与白蛋白的基础结构生物学相关研究及其医学意义之间的差距。事先已经确定了九种不同的药理上重要的药物类别，将在本项目的这一部分进行研究。这些类别是：1)止痛药(止痛药)，2)抗凝剂，3)麻醉药，4)尿酸(预防肾结石)，5)尿酸(痛风药物)，6)细胞抑制药，7)类固醇(激素)，8)维生素，9)抗生长素。然后通过EPR光谱和其他物理技术(如等温滴定量热法)表征了这些自旋标记药物与白蛋白的结合。此外，还表征了当配体结合时，来自脉冲EPR(DER)测量的白蛋白(人)可塑性的变化。这包括了解药物和脂肪酸同时结合的变构效应。然后，这项研究旨在探索(药物、脂肪酸)负载(人、牛)蛋白与人工膜的相互作用以及随之而来的蛋白质结构/可塑性的变化。这将通过EPR光谱方法和表面敏感红外反射吸收光谱(IRRAS)相结合来实现。这项提议的第二个大目标是了解六种不同哺乳动物蛋白的结构-功能关系，以及人类和动物蛋白在结构、可塑性和功能上的差异可能会出现。这一部分旨在将白蛋白的进化与溶液结构可塑性的差异联系起来。因此，建议用EPR跟踪进化：使用EPR光谱研究平台来表征白蛋白溶液结构，以找出在共同哺乳动物祖先的分化进化中，白蛋白表面的可塑性(如在HSA中所见)出现在哪里。然后研究了与HSA的序列同源性是显示这种可塑性所必需的，以及哪些氨基酸(主要位于蛋白质表面)对于从静态(BSA)转换到可塑性(HSA)是重要的。从这一点出发，然后描述可塑性差异如何转化为功能差异，例如在配体的摄取、释放或运输方面。最后，研究了白蛋白的翻译后修饰如何改变结构-功能关系。

Building on an EPR (electron paramagnetic resonance)-based research platform recently developed for the study of serum albumins, this proposal has two major objectives that are interconnected. The first goal is to understand structural plasticity and dynamics of serum albumin (human and animal, e.g. bovine) when binding small pharmaceutically active molecules and when interacting with a membrane. This will be achieved using EPR spectroscopy and will help bridging the gap between fundamental structural biology-related research on albumin and its medical implications. Nine different pharmacologically important classes of drugs have been identified beforehand that shall be studied in this part of the project. These classes are: 1) Analgetics (drugs for pain relief), 2) Anticoagulants; 3) Narcotics; 4) Uricostatics (prevention of kidney stones); 5) Uricosurics (gout medication); 6) Cytostatics; 7) Steroids (hormones); 8) Vitamins; 9) Antiauxins. The binding of these spin-labeled drugs to albumin by EPR spectroscopy and other physical techniques (e.g. isothermal titration calorimetry, ITC) is then characterized. Furthermore, changes in plasticity of albumins (human) as derived from pulse EPR (DEER) measurements when ligands are bound are characterized. This includes understanding allosteric effects of concurrent binding of drugs and fatty acids. Then the study aims at exploring interactions of (drug-, fatty-acid-)loaded (human, bovine) albumins with artificial membranes and the ensuing changes in structure/plasticity of the protein. This will be achieved by a combination of EPR spectroscopic methods and surface-sensitive infrared reflection-absorption spectroscopy (IRRAS). The second big aim of this proposal is to understand the structure-function relationship of six different mammal albumins and how differences in structure, plasticity and function of albumins (human and animal) may have arisen. This part thus aims at connecting the evolution of albumins with the difference in solution structural plasticity. The proposal hence is to 'follow evolution with EPR': Use the EPR-spectroscopic research platform for characterizing albumin solution structure to find out where in the divergent evolution from a common mammal ancestor the plasticity of albumin surface (as seen in HSA) has emerged. Then it is studied how much sequence identity with HSA is necessary to display this plasticity and which amino acids, mostly on the surface of the proteins, are important to switch from static (BSA) to plastic (HSA). Moving from this point, it is then characterized how differences in plasticity translate into functional differences, e.g. in uptake, release or transport of ligands. Finally, it is investigated how posttranslational modification of albumins alters the structure-function relationship.