Molecular Mechanisms, Pathways and Inhibition of Acetyl-Transfer Reactions

家长奖励R35GM118090的这一行政补充的目标是购买TwoMP MASS REFEYN公司的光度计用于分析大量不同的相关大分子组件 向家长奖致敬。在家长奖中研究的许多系统调节它们的生物学功能 通过形成大分子组件，这些组件的可靠表征是 对提取有关生物功能的信息和优化后续结构的样本是必不可少的 使用X射线结晶学或单颗粒(SP)低温电子显微镜进行表征。质量光度法是一种无需标记的 单分子溶液技术，使我们能够快速(每个样本几分钟)、准确和 相对较低的样品需求(~20 NM样品的~10μL)和几分钟的周转，分析 各种大小从30 kDa-5丙二醛不等的均相和杂相大分子组件，可产生 有关分子质量、低聚物之间的分布、化学计量以及动力学和解离的信息 在~20 PM到~500 NM范围内的常量。我们将使用TwoMP质谱仪来分析 包括蛋白质和核酸在内的大分子组装在各种缓冲和配位下 条件。定点突变体对大分子相互作用的影响也将用 质量光度学。此外，由于所需样品的浓度和体积极低 对于这项技术，我们将能够分析来自真核表达系统的样本，这些样本通常是 以低产量生产，否则无法使用其他生物物理方法进行分析。这些弥撒 光度分析也将在确定后续X射线的合适条件方面发挥关键作用 大分子及其络合物的结晶学和SP冷冻电子显微镜分析。特定的项目将 该仪器的好处包括对多蛋白蛋白和RNA乙酰转移酶的研究(SAS2/4/5， CAF1和Nat10)，N末端蛋白乙酰转移酶(NAT)调节(NAT复合体和核糖体)，以及 小分子乙酰基转移酶(ACLY、ACSS2、FASN、ACC1)。目前没有弥撒 校园内的光度计或类似仪器。该仪器将放置在专门的生物物理室 Marmorstein实验室和Marmorstein博士的家生化系内的设备室 生物物理学致力于在整个过程中正确安装和维护质量光度计 一辈子。

The goal of this administrative supplement of the parent award, R35GM118090, is to purchase a TwoMP Mass Photometer from REFEYN for the analysis of a large number of diverse macromolecular assemblies relevant to the parent award. Many of the systems under study in the parent award mediate their biological functions through the formation of macromolecular assemblies, and a reliable characterization of these assemblies is essential for drawing information about biological function and for optimizing samples for subsequent structural characterization using X-ray crystallography or single particle (SP) cryo-EM. Mass Photometry is a label-free single molecule solution technique that will allow us to rapidly (minutes per sample), accurately, and with relatively low sample requirement (~10 μL of a ~20 nM sample) and several-minute turnaround, analyze various homomeric and heteromeric macromolecular assemblies ranging in size from 30 kDa – 5 MDa, yielding information about molecular mass, distribution among oligomers, stoichiometry, and kinetics and dissociation constants in the range of ~20 pM to ~500 nM. We will use the TwoMP Mass Photometer to analyze macromolecular assemblies including proteins and nucleic acids under a variety of buffer and liganded conditions. The effect of site directed mutants on macromolecular interactions will also be evaluated using Mass Photometry. In addition, because of the exceedingly low concentration and volume of sample required for the technology, we will be able to analyze samples from eukaryotic expression systems that are typically produced in low yield that could not otherwise be analyzed using other biophysical approaches. These Mass Photometry analyses will also play a key role in identifying suitable conditions for subsequent X-ray crystallographic and SP cryo-EM analysis of macromolecules and their complexes. Specific projects that will benefit from the instrumentation include studies on multiprotein protein and RNA acetyltransferases (Sas2/4/5, Caf1 and Nat10), N-terminal protein acetyltransferase (NAT) regulation (NAT complexes and ribosomes), and small molecule acetyl-transfer enzymes (ACLY, ACSS2, FASN, ACC1). There is currently no Mass Photometer or comparable instrument on campus. The instrument will be located in a dedicated biophysical equipment room within the Marmorstein laboratory and Dr. Marmorstein’s home Department of Biochemistry and Biophysics is committed to the proper installation and maintenance of the Mass Photometer throughout its lifetime.