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Chemically engineered bilayers for cryoEM imaging of membrane proteins in continuous membranes

用于连续膜中膜蛋白冷冻电镜成像的化学工程双层

基本信息

批准号：
10091731
负责人：
Aviv Paz
金额：
$ 29.1万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
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：
2019-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10091731
关键词：
3-Dimensional Address Affinity Biochemical Biological Biological Models Caliber Carbon Cell membrane Cell physiology Cells Chemical Engineering Chemicals Cryoelectron Microscopy Crystallization Data Data Analyses Destinations Detergents Development Electrons Engineering Environment Event Face Film Financial compensation Generations Heterogeneity Ice Image Inositol Ion Channel Label Laboratories Ligands Lipid Bilayers Lipids Membrane Membrane Lipids Membrane Proteins Methods Microscopic Miniaturization Modeling Molecular Conformation Phase Physiological Plant Roots Play Post-Translational Protein Processing Procedures Process Proteins Publishing Recombinants Resolution Role Signal Transduction Structure Surface System Technology Testing Thick Tissues Vesicle Visualization Work base chemical group density electron crystallography improved membrane reconstitution milligram mimetics nanodisk nanometer nanoscale new technology particle protein complex receptor reconstitution reconstruction success technology development unilamellar vesicle

项目摘要

Cells interact with their environments through membrane proteins. Structural and functional studies of membrane proteins are thus very important. Structure determination of eukaryotic membrane proteins in membrane however remains difficult despite substantial progresses. Part of the challenge comes from the fact that many eukaryotic membrane proteins undergo a complicated intracellular maturation process and carry different post-translational modifications before reaching their final destinations. Current high throughput crystallization and cryoEM single particle reconstruction are largely carried out with proteins in detergents, or in membrane-mimetic systems such as bicelles, nanodiscs, lipid-cubic phases or amphipols, where there are still significant differences in comparison with a native membrane. New technologies are needed to overcome these problems. We propose here to develop two new technologies for cryoEM study of membranes proteins in continuous membrane using type 1 IP3 receptor (IP3R) as a working model. The premise of these two methods is partly based on our recent work of a chemical engineering procedure that is suitable for functionalizing nanometer-thick carbon films and of a bead-supported spherical unilamellar membrane (bSUM) system that allows the generation of stable giant unilamellar vesicles. With milligram amounts of IP3R proteins, we will produce a nanometer-bSUM (nm-bSUM) and a carbon-supported planar unilamellar membrane (cPUM). These two systems will be prepared for the cryoEM visualization of the IP3Rs in continuous membrane where the proteins are fully immersed in a lipid bilayer, and will allow us to resolve the receptor structure from images of membrane-integrated molecules. Images of the receptors in nm-bSUMs will be used for random spherically constrained (RSC) reconstruction. Receptors in cPUMs will be imaged at high tilt angles for 3D reconstruction with corrections for changes in defocus levels across the imaging field. Both methods will rely on chemical engineering and membrane reconstitution at the nanometer scale and will result in efficient unidirectional insertion of membrane proteins at sub-nM concentrations, which will be particularly beneficial for selecting specifically labeled mature functional membrane proteins or enriching low-abundance membrane protein complexes at sub-nM concentrations. Results of the proposed studies will create new windows of opportunities for cryo-EM study of various membrane protein complexes in membrane and for using nanoscale membrane systems in other bioanalytical or biomedical applications. ! 1!

细胞通过膜蛋白与环境相互作用。的结构和功能研究