PROTEIN BEHAVIOR AT SILICONE-WATER INTERFACES

硅-水界面上的蛋白质行为

• 批准号: 2634719
• 负责人: VIOLA VOGEL
• 金额: $ 9.96万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634719
• 关键词: acidity /alkalinity; acrylamides; adsorption; bilirubin; biomaterial interface interaction; chromophore; coagulation factor IX; complement; conformation; fibronectins; fluorescence microscopy; fluorescence spectrometry; intermolecular interaction; iodine; monoclonal antibody; oils; organosilicon compound; phosphatidylcholines; protein structure; serum albumin; thermodynamics; time resolved data; tryptophan; water

We propose to study conformational alternations of proteins adsorbed from solutions to water/silicone oil interfaces with the aim of assessing on the molecular level how silicone based implants, such as leaking breast implants, cause foreign body reactions. A long standing hypothesis is that the observed immunological responses do not originate from the silicone implants themselves, since silicones are characterized as chemically inert, but via proteins that undergo conformational alterations on surface adsorption. Protein adsorption to a synthetic surface occurs immediately after exposure; it hence constitutes the primary response. Little is known regarding protein conformation in the adsorbed state, not only on silicone oil surfaces but in general, since three-dimensional protein structures within adsorbate films can hardly be determined directly with monolayer sensitivity. It is therefore proposed to measure a set of physiochemical parameters for model proteins adsorbed to water/silicone interfaces, where each parameter probed reflects another feature of the conformationally altered protein, and then to compare these parameters for the same model protein adsorbed to other, far better characterized interfaces. It is assumed that if adsorbed proteins are similar in a sufficient number of physicochemical parameters they will express similar functional behavior. Experimentally, advantage is taken from the fact that silicone oils spread at the air/water interface. Protein behavior on adsorption to the water/silicone oil interface will hence directly be compared to that found at water/air and membrane mimetic water/lipid interfaces. This adsorption studies to all these interfaces will be done in a Langmuir trough. The uniqueness of this approach is that these three interfaces will be probed under similar conditions and by the same set of techniques which include measurements of surface potential, fluorescence spectroscopy and microscopy, time-resolved fluorescence anisotropy decay. The molecular ordering will be probed by a new powerful nonlinear optical laser technique, optical second harmonic generation. The model proteins are human serum albumin and human fibronectin. We will study their conformations at model interfaces and how they respond to solvent perturbation and pH (Experiment 1). The location of the tryptophan residues is deferred from probing their polar environment (Experiment 2), and their accessibility to aqueous quenches (Experiment 3). The conformational alterations will be related to the expressed function by studying the ability of surface adsorbed proteins to bind to ligands (Experiment 4). Information on the dynamics of the three- dimensional protein structure will be gained from time-resolved fluorescence anisotropy decay studies (Experiment 5). The texture of the adsorbed protein film will be imaged by fluorescence microscopy (Experiments 6), and the biological activity of the conformationally altered proteins will be assessed by studying their interaction with complement proteins and monoclonal antibodies (Experiment 7). The ultimate goal is to provide insight on the molecular level whether and how conformationally altered surface proteins may act indirectly as initiators of inflammatory responses.

我们建议研究从吸附的蛋白质的构象变化 水/硅油界面的解决方案，旨在评估 分子水平如何基于硅树脂的植入物，例如乳房渗漏 植入物，引起异物反应。 一个长期存在的假设是 观察到的免疫反应并非源自 硅胶植入物本身，因为硅胶的特点是 化学惰性，但通过经历构象的蛋白质 表面吸附的改变。 蛋白质对合成物的吸附 暴露后立即出现表面；因此它构成了 主要反应。 关于吸附状态下的蛋白质构象知之甚少，不知道 仅在硅油表面上，但在一般情况下，因为三维 吸附膜内的蛋白质结构很难确定 直接与单层敏感性。 因此建议测量 吸附模型蛋白质的一组理化参数 水/硅界面，其中探测的每个参数都反映另一个参数 构象改变的蛋白质的特征，然后进行比较 这些参数对于同一模型蛋白质吸附到其他蛋白质来说要好得多 特征化的接口。 假设如果吸附的蛋白质 它们在足够数量的物理化学参数上相似 表达相似的功能行为。 实验上，硅油的优点是 在空气/水界面处传播。 蛋白质吸附行为 因此，水/硅油界面将直接与该界面进行比较 发现于水/空气和膜模拟水/脂质界面。 这 所有这些界面的吸附研究将在 Langmuir 中进行 槽。 这种方法的独特之处在于这三个接口 将在相似的条件下并采用相同的技术进行探测 其中包括表面电位、荧光的测量 光谱学和显微镜，时间分辨荧光各向异性衰减。 分子排序将通过一种新的强大的非线性光学来探测 激光技术，光学二次谐波发生。 模型蛋白是人血清白蛋白和人纤连蛋白。 我们 将研究它们在模型界面的构象以及它们如何响应 溶剂扰动和 pH 值（实验 1）。 的位置 色氨酸残基被推迟探测其极地环境 （实验 2），以及它们对水性淬火的可及性（实验 3）。 构象改变将与所表达的 通过研究表面吸附蛋白结合的能力来发挥功能 配体（实验4）。 三者动态信息 三维蛋白质结构将从时间分辨中获得 荧光各向异性衰减研究（实验 5）。 的质地 吸附的蛋白质膜将通过荧光显微镜成像 （实验6），以及构象的生物活性 改变的蛋白质将通过研究它们与 补体蛋白和单克隆抗体（实验 7）。 这 最终目标是在分子水平上提供关于是否和 构象改变的表面蛋白如何间接发挥作用 炎症反应的引发剂。