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.

我们建议研究吸附蛋白质的构象变化， 水/硅油界面的解决方案，旨在评估 在分子水平上， 植入物会引起异物反应 一个长期存在的假设是， 所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，但通过蛋白质， 改变表面吸附。 蛋白质吸附到合成 表面在暴露后立即发生;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， 仅在硅油表面上，但一般来说，由于三维 吸附物膜内的蛋白质结构很难确定 直接与单层灵敏度。 因此，建议测量 一组物理化学参数的模型蛋白质吸附到 水/硅胶界面，其中探测的每个参数反映另一个参数 改变蛋白质构象的特征，然后比较 这些参数对于同一模型蛋白质吸附到其它的， 特征接口。 据推测，如果吸附的蛋白质 在足够数量的物理化学参数方面类似，它们将 表达类似的功能行为。 在实验上，优势在于硅油 在空气/水界面上扩散。 蛋白质吸附行为 因此，水/硅油界面将直接与 发现在水/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朗缪尔 槽。 这种方法的独特之处在于这三个接口 将在类似的条件下用同样的技术进行探测 包括测量表面电位、荧光 光谱和显微镜，时间分辨荧光各向异性衰减。 分子有序性将被一种新的强非线性光学方法所探测 激光技术，光学二次谐波产生。 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上的构象以及它们的反应 溶剂扰动和pH（实验1）。 的位置 色氨酸残基不能探测它们的极性环境 （实验2），以及它们对水性猝灭剂的可及性（实验3）。 3）。 构象改变将与表达的 通过研究表面吸附的蛋白质结合到 配体（实验4）。 关于这三个国家的动态的信息- 蛋白质的三维结构将从时间分辨 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），和构象上的生物活性。 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）。 的 最终目标是提供分子水平上的洞察力， 构象改变的表面蛋白如何间接地作为 引发炎症反应。