Tools to Connect Protein Conformations, Dynamics, and Associations

连接蛋白质构象、动力学和关联的工具

基本信息

批准号：
8354224
负责人：
DANIEL SCHWARTZ
金额：
$ 18.01万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8354224
关键词：
Address Adhesions Adjuvant Adopted Adsorption Affect Affinity Area Behavior Binding Biocompatible Biocompatible Materials Biological Biological Models Biomedical Engineering Biomedical Technology Biosensing Techniques Biosensor Cell Adhesion Characteristics Chemistry Color Complex Crowding Diagnostic Diffusion Electrostatics Energy Transfer Environment Equipment Extracellular Matrix Proteins Fibronectins Film Fluorescence Microscopy Fluorescence Resonance Energy Transfer Goals Health Heterogeneity Human Hydrocarbons Hydrophobic Interactions Individual Label Laboratories Lateral Lead Measures Mediating Medical Membrane Proteins Methods Microscopy Modeling Molecular Molecular Conformation Molecular Structure Peptides Polyethylene Glycols Population Preparation Process Property Protein Conformation Proteins Research Resistance Role Safety Silicon Dioxide Solid Surface Techniques Testing Time Vaccines Work aqueous base biomaterial compatibility design implantable device improved interfacial molecular dynamics monomer novel prevent protein aggregation protein protein interaction protein structure receptor binding research study residence response single molecule surface coating therapeutic protein tissue culture tool vaccine efficacy

项目摘要

DESCRIPTION (provided by applicant): The overall objective of this work is to develop novel single molecule microscopy methods that will enable mechanistic studies of the ways in which surface chemistry affects adsorbed protein conformation and intermolecular associations. Although resistance to protein adsorption is often cited as necessary for a particular application (biosensing, biocompatibility, etc.), even the most protein- resistant surfaces permit some protein adsorption. Therefore, this work will test the hypothesis that vicinal surface chemistry indirectly affects protein behavior after adsorption to influence the propensity for intermolecular associations (binding, aggregation, etc.). A mechanistic understanding of post- adsorptive protein behavior and the ability of the surface to mediate this behavior will ultimately lead to better surface coatings for a variety of biomedical technologies. As a relevant and convenient model system, fluorescently-labeled fibronectin (Fn) will be studied on model biocompatible surfaces as well as surfaces that specifically probe electrostatic, hydrophilic and hydrophobic interactions. This work will use single-molecule fluorescence microscopy techniques at the solid-aqueous interface that are capable of simultaneously measuring the molecular conformation of an individual Fn molecule and tracking its effect on dynamic processes such as adsorption, diffusion, desorption, aggregation, and receptor binding. Resonance energy transfer between Fn labels will be used to probe molecular conformation. The first specific aim will examine the ability of different surfaces to influence Fn conformation and the subsequent effect this has on Fn surface affinity and diffusion. Building on this understanding, the second aim will address protein-protein interactions for their propensity to form a stable protein film. The surfac is expected to indirectly influence film formation through the mobility of proteins on the surface and their propensity for cluster formation, properties that likely depend on Fn conformation. PUBLIC HEALTH RELEVANCE: Interactions between surfaces and proteins have widespread relevance to human health, affecting medical diagnostics, therapeutic protein stability, vaccine efficacy/safety, and biomaterial design. A common technological goal involves the preparation of "protein- resistant" surfaces. However, the mechanisms of protein resistance remain elusive, and even the most biocompatible surfaces fail to prevent adhesion entirely. To resolve this inconsistency, this work will develop new microscopy techniques to test the hypothesis that biocompatible surfaces permit adhesion of proteins that are unlikely to trigger an adverse response and may also influence their post-adhesion behavior to prevent them from adopting unwanted behavior. A more complete understanding of the role of the surface in biocompatibility will lead to the design of improved surfaces for biosensors, implanted devices and equipment for biological experimentation.

描述（由申请人提供）：这项工作的总体目的是开发新型的单分子显微镜方法，该方法将对表面化学影响吸附蛋白构象和分子间关联的方式进行机械研究。尽管对于特定应用（生物传感，生物相容性等），通常会引用对蛋白质吸附的耐药性，但即使是最耐蛋白质的表面也允许一些蛋白质吸附。因此，这项工作将检验以下假设：替代表面化学在吸附后间接影响蛋白质行为，以影响分子间的倾向关联（绑定，聚合等）。对后吸附蛋白行为的机械理解以及表面介导这种行为的能力最终会为各种生物医学技术提供更好的表面涂层。作为一个相关且方便的模型系统，将在模型的生物相容性表面以及专门探测静电，亲水性和疏水性相互作用的表面上研究荧光标记的纤连蛋白（FN）。这项工作将在固体界面上使用单分子荧光显微镜技术，这些界面能够同时测量单个FN分子的分子构象并跟踪其对动态过程的影响，例如吸附，扩散，降解，脱附，聚集，聚集和受体结合。 FN标签之间的共振能传递将用于探测分子构象。第一个具体目的将检查不同表面影响FN构象的能力，以及随后对FN表面亲和力和扩散产生的影响。在这种理解的基础上，第二个目标将解决蛋白质 - 蛋白质相互作用，以形成稳定的蛋白质膜。预期表面有望通过蛋白质在表面上的迁移率及其群集形成的倾向，可能取决于FN构象的特性，从而间接影响膜的形成。公共卫生相关性：表面和蛋白质之间的相互作用与人类健康具有广泛的相关性，影响医学诊断，治疗蛋白质稳定性，疫苗功效/安全性和生物材料设计。一个共同的技术目标涉及制备“抗蛋白质”表面。但是，蛋白质耐药性的机制仍然难以捉摸，甚至最生物相容性的表面也无法完全防止粘附。为了解决这种不一致，这项工作将开发新的显微镜技术，以检验以下假设：生物相容性表面允许蛋白质的粘附，这些蛋白质不太可能引发不良反应并可能影响其粘附后行为以防止它们采用不必要的行为。对表面在生物相容性中的作用的更全面了解将导致设计改进的生物传感器，植入设备和生物学实验设备的表面。