PROTEIN BEHAVIOR AT SILICONE-WATER INTERFACES

硅-水界面上的蛋白质行为

• 批准号: 2022709
• 负责人: VIOLA VOGEL
• 金额: $ 9.77万
• 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 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022709
• 关键词: acidity /alkalinity; acrylamides; bilirubin; biomaterial interface interaction; coagulation factor IX; conformation; fluorescence spectrometry; thermodynamics

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.

我们建议研究吸附在蛋白质上的构象变化。 水/硅油界面的解决方案，目的是评估 分子水平如何植入硅胶假体，如乳房渗漏 植入物会引起异物反应。一个长期存在的假设是 观察到的免疫反应不是源于 硅胶植入物本身，因为硅酮的特征是 化学上是惰性的，但通过蛋白质的构象 表面吸附的变化。蛋白质在人工合成材料上的吸附 表面在曝光后立即发生；因此，它构成 主要反应。 关于蛋白质在吸附状态下的构象，我们知之甚少 仅限于硅油表面，但一般情况下，因为三维 吸附膜内的蛋白质结构很难确定。 直接以单分子层灵敏度。因此，建议测量 一组模型蛋白质的物理化学参数 水/硅胶界面，其中每个探测参数反映另一个参数 构象改变的蛋白质的特征，然后比较 这些参数对于相同模型的蛋白质吸附到其他模型中，要好得多 特征化的接口。假设如果吸附的蛋白质是 在足够数量的物理化学参数上相似，它们将 表达类似的功能行为。 从实验上讲，硅油的优势在于 在空气/水界面扩散。蛋白质在土壤中的吸附行为 因此，水/硅油界面将直接与 发现于水/空气和膜模拟水/脂界面。这 对所有这些界面的吸附研究将在朗缪尔进行 低谷。这种方法的独特之处在于，这三个接口 将在类似条件下通过相同的一套技术进行探测 其中包括表面电位、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大的非线性光学所探测 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维连接蛋白。我们 将研究它们在模型界面上的构象以及它们如何响应 对溶剂扰动和pH的影响(实验1)。的位置。 色氨酸残留物推迟了对其极地环境的探测 (实验2)，以及它们对水淬火的可及性(实验 3)。构象变化将与表达的 通过研究表面吸附的蛋白质结合的能力来发挥作用 配体(实验4)。关于三个国家的动态的信息- 三维蛋白质结构将通过时间分辨获得 荧光各向异性衰减研究(实验5)。它的质地 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蛋白质的相互作用进行评估 补体蛋白和单抗(实验7)。这个 最终目标是在分子水平上提供洞察是否和 构象改变的表面蛋白如何间接作用于 炎症反应的发起者。