Real Time (RT) In-cell NMR technology to study protein interactions in live cells

实时 (RT) 细胞内 NMR 技术用于研究活细胞中的蛋白质相互作用

• 批准号: 9912182
• 负责人: ALEXANDER SHEKHTMAN
• 金额: $ 30.79万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912182
• 关键词: Affect; Affinity; Binding; Biochemical; Biological; Biological Assay; Biology; Cell Survival; Cells; Cellular Structures; Cellular biology; Chemistry; Communities; Complex; Computing Methodologies; Cryopreserved Cell; Culture Media; Diabetes Mellitus; Disease; Drug Compounding; Drug Screening; Electron Microscopy; Environmental Risk Factor; Eukaryotic Cell; Glean; Goals; Grant; Growth; Hour; Human; Human Cell Line; In Situ; In Vitro; Individual; Infrastructure; Laboratories; Ligands; Mammalian Cell; Methods; Microscopy; Modeling; Modernization; Molecular Weight; Monitor; NMR Spectroscopy; Nature; New York; Perfusion; Pharmaceutical Preparations; Prokaryotic Cells; Property; Protein Analysis; Protein Biochemistry; Proteins; Protocols documentation; RNA; Readiness; Relaxation; Research; Resolution; Signal Transduction; Stimulus; Structure; Structure-Activity Relationship; Techniques; Technology; Testing; Time; Time Study; Treatment Efficacy; Tube; Universities; Work; biological systems; data acquisition; experimental study; in vivo; instrumentation; interest; light microscopy; meetings; nanomolar; new technology; protein complex; protein expression; protein protein interaction; protein structure; protein structure function; structural biology; therapeutic target; tool

Real Time (RT) In-cell NMR technology to study protein interactions in live cells Alexander Shekhtman1 1Department of Chemistry, University at Albany, State University of New York, Albany, NY 12222 ABSTRACT: Since intracellular changes induced by stimuli can take hours to fully manifest into detectable signals, one of the overarching goals of modern biology is to understand temporal protein structure-function relationships at atomic resolution within the complexity of a live cell. In-cell solution NMR spectroscopy is an important step towards this goal but is limited by the long data acquisition times and the static nature of in-cell NMR experiments that provide snapshots rather than continuous monitoring of time dependent changes in protein structure. In addition, weak quinary interactions between the protein of interest and intracellular components, particularly RNAs, which are omnipresent in live cells, result in a dramatic increase in the apparent in-cell molecular weight and render all but a few proteins invisible by standard in-cell NMR approaches. There are currently no structural biology tools to characterize time dependent protein interactions in live cells at atomic resolution even though these interactions affect protein physicochemical properties, protein-protein, protein-ligand, and protein-drug binding. We showed that the combination of protein deuteration and NMR experiments using optimized transverse relaxation allowed us to obtain in-cell NMR spectra of previously invisible proteins. The goal of this project is to develop real time (RT) in-cell NMR technology to characterize protein interactions in situ over a long (more than a day) period of time at atomic resolution inside live prokaryotic and eukaryotic cells. We will apply this technology to study how exogenous and endogenous challenges to cells result in specific temporal changes in protein structure. We will build the infrastructure to make this new technology available to the scientific community. We expect that the technology will be critical to bridge the gap between in vitro and in-cell protein biochemistry, which is an absolute requirement to understand cell biology and to develop effective therapeutics against protein targets.

真实的时间（RT）细胞内NMR技术用于研究活细胞中的蛋白质相互作用 亚历山大谢克特曼1 1纽约州立大学奥尔巴尼分校化学系，奥尔巴尼，NY 12222 摘要：由于刺激诱导的细胞内变化可能需要数小时才能完全表现出来， 现代生物学的首要目标之一是了解时间蛋白 在一个活细胞的复杂性内的原子分辨率的结构-功能关系。细胞内解决方案 核磁共振光谱是实现这一目标的重要一步，但受到长数据采集时间的限制 以及提供快照而不是连续的细胞内NMR实验的静态性质 监测蛋白质结构的时间依赖性变化。此外，弱五元相互作用之间 目的蛋白质和细胞内组分，特别是RNA，其在肝脏中无所不在 细胞，导致细胞内表观分子量的急剧增加， 蛋白质不可见的标准细胞内NMR方法。目前还没有结构生物学工具来 以原子分辨率表征活细胞中的时间依赖性蛋白质相互作用， 相互作用影响蛋白质物理化学性质、蛋白质-蛋白质、蛋白质-配体和蛋白质-药物 约束力我们表明，蛋白质氘化和NMR实验的组合，使用优化的 横向弛豫使我们能够获得以前不可见的蛋白质的细胞内NMR谱。目标 该项目的主要目的是开发真实的时间（RT）细胞内NMR技术，以表征蛋白质相互作用， 在活的原核细胞内以原子分辨率长时间（超过一天）原位观察， 真核细胞我们将应用这项技术来研究如何外源性和内源性的挑战， 细胞导致蛋白质结构的特定时间变化。我们将建立基础设施， 科学界可利用的新技术。我们预计这项技术将对 弥合体外和细胞内蛋白质生物化学之间的差距，这是绝对的要求， 了解细胞生物学并开发针对蛋白质靶点的有效疗法。