Nanomechanical imaging of protein dynamics via programmable DNA interactions

通过可编程 DNA 相互作用进行蛋白质动力学纳米力学成像

基本信息

批准号：
10020421
负责人：
Ozgur Sahin
金额：
$ 32万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10020421
关键词：
3-Dimensional Address Adoption Area Atomic Force Microscopy Bacteriorhodopsins Binding Binding Proteins Biological Models Biological Process Biomedical Research Biotin Calibration Chemicals Color Complex Crystallography DNA DNA Probes DNA Sequence Data Analyses Detection Development Disease Electron Microscopy Enzymes Equilibrium Fluorescence Microscopy Goals Image Image Analysis Image Enhancement Imaging technology Individual Institution Kinetics Label Membrane Membrane Proteins Methods Microscope Microscopy Molecular Multienzyme Complexes NMR Spectroscopy Performance Pharmaceutical Preparations Physiological Protein Dynamics Proteins Protocols documentation RNA Research Resolution Series Shapes Signal Transduction Site Specificity Speed Streptavidin Structural Protein Structure System Techniques Technology Testing Validation X-Ray Crystallography base cantilever chemical group design flexibility image reconstruction imaging platform improved innovation nanomechanics new technology programs protein complex reconstruction single molecule small molecule structural biology technology development tool

项目摘要

PROJECT SUMMARY The proposed research will address challenges in imaging dynamic structures of proteins and protein complexes in solution. Fluorescence microscopy is currently the dominant technique for chemically specific imaging in solution. Although super-resolution techniques have dramatically pushed the resolution limits of fluorescence microscopy, it is still a major challenge to obtain images of single-molecules and molecular complexes with resolutions comparable to electron microscopy and X-ray crystallography. Our proposed research will develop an atomic force microscopy (AFM) platform that harnesses short-lived DNA/DNA interactions to achieve imaging and three-dimensional reconstruction of chemical groups within individual proteins, multi-enzyme complexes, and complexes between proteins, RNA and DNA with near atomic resolution and in physiologically relevant conditions. Enhancing the imaging speed will enable observation of structural transitions. These capabilities are enabled by a series of innovations: (1) the use of short fragments of DNA as imaging labels, wherein the sequence of DNA encodes color information, (2) the use of short-lived, far from equilibrium interactions that boost resolution and chemical specificity, and (3) the use of specially designed T-shaped atomic force microscopy cantilevers that allow detecting short-lived interactions. The resulting microscopy platform will have a broad impact across many biomedical fields by providing structural information about proteins and protein complexes that are difficult to study via the current methods.

项目概要拟议的研究将解决蛋白质和蛋白质动态结构成像的挑战溶液中的配合物。荧光显微镜是目前化学特异性分析的主导技术溶液中成像。尽管超分辨率技术极大地突破了分辨率极限荧光显微镜，获得单分子和分子图像仍然是一个重大挑战复合物的分辨率可与电子显微镜和 X 射线晶体学相媲美。我们提出的研究将开发一个利用短寿命 DNA/DNA 的原子力显微镜 (AFM) 平台相互作用以实现个体内化学基团的成像和三维重建蛋白质、多酶复合物以及蛋白质、RNA 和 DNA 之间具有近原子结构的复合物分辨率和生理相关条件。提高成像速度将能够观察到结构转变。这些功能是通过一系列创新实现的：（1）短片段的使用 DNA作为成像标签，其中DNA序列编码颜色信息，(2)使用短寿命、远离提高分辨率和化学特异性的平衡相互作用，以及（3）使用特殊的设计了 T 形原子力显微镜悬臂，可以检测短暂的相互作用。这由此产生的显微镜平台将通过提供结构性的结果对许多生物医学领域产生广泛的影响有关通过当前方法难以研究的蛋白质和蛋白质复合物的信息。