Diffusing Colloidal Probe Measurements of Protein and Synthetic Macromolecule Interactions

蛋白质和合成大分子相互作用的扩散胶体探针测量

• 批准号: 0829226
• 负责人: Michael Bevan
• 金额: $ 7.4万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-02 至 2009-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0829226&HistoricalAwards=false
• 关键词: Diffusing; Colloidal; Probe; Measurements; Protein

ABSTRACT Diffusing Colloidal Probe Measurements of Protein and Synthetic Macromolecule Interactions This proposal is concerned with the application of a conceptually new method, Diffusing Colloidal Probe Microscopy (DCPM), to directly and nonintrusively measure kT interactions of surface immobilized proteins and synthetic macromolecules. Ensembles of freely diffusing colloids will be employed as ultra-sensitive probes to measure energy vs. separation dependent potentials between proteins covalently attached to colloids and planar surfaces. By combining evanescent wave and video microscopy techniques, DCPM will monitor three dimensional Brownian excursions of protein decorated colloids as they sample positions normal and parallel to homogeneous, heterogeneous, and patterned substrates also bearing covalently attached and oriented proteins. Because diffusing probes sample spatial positions according to their relative energies, time and ensemble averaged excursions of probes can be interpreted using statistical mechanical analyses in terms of net potentials of superimposable non-specific (colloidal, macromolecular) and specific (residues, geometry) contributions. Consistent with the nanotechnology paradigm, DCPM will interrogate protein and synthetic macromolecule interactions by exploiting natural gauges for time (a2/D), energy (kT), force (fN), and length (nm) associated with diffusing colloidal probes. The broad aim of the proposed research is to gain fundamental insights into protein-protein and protein-synthetic macromolecule interactions important to non-specific adsorption of proteins on synthetic surfaces and for interpreting specific binding events measured on protein arrays. Specific experimental and modeling objectives are focused on using DCPM to measure interactions of proteins attached and oriented on sub-micron colloids and micron scale surface patterns. The first task of the proposed work is to covalently attach/orient proteins onto gold colloids and either gold or silica surfaces (purified and characterized proteins provided by collaborator). Building on preliminary DCPM results for physisorbed proteins and synthetic macromolecules, the second task is to measure interactions of covalently attached/oriented proteins with various mutations and conformations to allow for deconvolution of non-specific and specific contributions to net potentials. The last task is to measure non-specific and specific interactions on a model array consisting of patterned regions with covalently attached/oriented proteins and regions of physisorbed synthetic macromolecules. Successful completion of proposed objectives will provide new fundamental insights into applications including biofouling and protein array diagnostics, and in the process, will demonstrate a new technology (DCPM) to directly and sensitively quantify weak interactions controlling non-covalent, equilibrium binding of proteins and their non-specific interactions with synthetic macromolecules. The intellectual merit of the proposed research is broadly related to the fundamental knowledge that will be gained in integrating nanoscale synthetic systems and materials of biological origin. Experimental objectives will produce unique, direct measurements of non-specific and specific protein-protein and protein-synthetic macromolecule interactions on energy (kT) and length (nm) scales relevant to equilibrium binding and adsorption. Modeling objectives will interpret/predict multi-scale non-specific and specific contributions to the net interaction of protein conjugated diffusing colloidal probes and surfaces bearing proteins and synthetic macromolecules. Educational objectives will involve using visual content generated in the proposed research in courses and outreach activities. In terms of classroom teaching, research images and videos will be incorporated into undergraduate and graduate colloid/polymer elective courses and an undergraduate thermodynamics core course. In terms of outreach, content involving optical microscopy of colloids will be adapted for use in programs for 7-12 grade teachers and students primarily through the PI's participation in an existing NSF center for learning and teaching. The broader impacts of this proposal involve exploiting visual research content to provide immersive educational experiences for students at all levels that minimizes boundaries between authentic and guided inquiry.

抽象扩散蛋白质和合成大分子相互作用的胶体探针测量该建议与概念上的新方法的应用有关，从而扩散了胶体探针显微镜（DCPM），以直接和非投入性地测量固定化蛋白质和合成大型巨质菌群的KT相互作用。自由扩散胶体的集合将用作超敏感探针，以测量与胶体和平面表面共同附着的蛋白质之间的蛋白质与分离依赖性电位。通过将evaneScent波和视频显微镜技术相结合，DCPM将监测蛋白质装饰的胶体的三维布朗游览，因为它们采样了正常且平行于同质，异构和图案的底物的样品位置，还具有共同的附着和方向的蛋白质。由于探针扩散根据样品空间位置根据其相对能量的相对能量，因此可以使用统计机械分析来解释探针的平均探针，以叠加的非特异性非特异性（胶体，大分子）和特定（残基，几何）的贡献来解释。与纳米技术范式一致，DCPM将通过利用自然仪表（a2/d），能量（kt），力（fn）和长度（NM）来审问蛋白质和合成大分子相互作用，与扩散胶体探针相关。拟议研究的广泛目的是获得对蛋白质 - 蛋白质和蛋白质合成的大分子相互作用的基本见解，这对蛋白质对合成表面的非特异性吸附至关重要，并解释在蛋白质阵列上测得的特定结合事件。特定的实验和建模目标的重点是使用DCPM测量在亚微米胶体和微米尺度表面模式上附着和定向的蛋白质的相互作用。拟议的工作的第一个任务是共价连接/定向蛋白在金胶体和金或二氧化硅表面上（合作者提供的纯化和表征蛋白质）。在初步的DCPM基础上，物理蛋白和合成大分子的结果是，第二任务是测量具有各种突变和构象的共同附着/定向蛋白的相互作用，以使非特异性和特定贡献对净电位进行反卷积。最后的任务是在模​​型阵列上测量非特定和特定的相互作用，该模型阵列由具有共价/定向/定向的蛋白质以及物理学的合成大分子的区域和区域组成。成功完成拟议的目标将为包括生物污染和蛋白质阵列诊断等应用提供新的基本见解，在此过程中，将展示一种新技术（DCPM），以直接且敏感地量化弱的弱相互作用，以控制非共价，平衡，蛋白质及其与合成千数质量的非特异性相互作用。拟议研究的智力优点与将纳米级合成系统和生物学来源材料整合在一起的基本知识广泛有关。实验目标将对与平衡结合和吸附相关的能量（KT）和长度（NM）尺度上的非特异性和特异性蛋白质 - 蛋白质和蛋白质合成的大分子相互作用产生独特的直接测量。建模目标将解释/预测对蛋白质共轭扩散的胶体探针和带有蛋白质和合成大分子的表面的多尺度非特异性和特定贡献。教育目标将涉及使用在课程和外展活动中提出的研究中产生的视觉内容。在课堂教学方面，研究图像和视频将纳入本科和研究生胶体/聚合物选修课程以及本科热力学核心课程。在外展方面，涉及胶体光学显微镜的内容将用于7-12年级教师和学生的课程中，主要是通过PI参与现有的NSF学习和教学中心。该建议的更广泛的影响涉及利用视觉研究内容，为各级学生提供沉浸式的教育经验，以最大程度地减少真实和指导性询问之间的界限。