Molecular Mechanisms, Pathways and Inhibition of Acetyl-Transfer Reactions

本行政补充的母公司奖励，R35GM118090的目标是购买一个TwoMP质量 REFEYN的光度计，用于分析大量不同的大分子组装体， 家长奖。许多系统正在研究中的父母奖调解其生物功能 通过形成大分子组装体，这些组装体的可靠表征是 对于提取有关生物功能的信息和优化用于随后结构分析的样品至关重要。 使用X射线晶体学或单颗粒（SP）冷冻EM进行表征。质量光度法是一种无标签 单分子溶液技术，这将使我们能够快速（每个样品分钟），准确， 相对较低的样品要求（约10 μ L的约20 nM样品）和几分钟的周转时间， 大小范围为30 kDa-5 MDa的各种同聚和异聚大分子组装体， 有关分子量、低聚物分布、化学计量、动力学和解离的信息 在约20 pM至约500 nM范围内的恒定。我们将使用TwoMP质量光度计来分析 包括蛋白质和核酸在内的大分子组装体在各种缓冲液和配体下， 条件定点突变体对大分子相互作用的影响也将使用 质谱光度法。此外，由于所需样品的浓度和体积非常低， 对于这项技术，我们将能够分析来自真核表达系统的样本， 以低产量产生，否则无法使用其他生物物理方法进行分析。这些大规模 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 晶体学和SP cryo-EM分析的大分子及其复合物。具体项目将 仪器的益处包括对多蛋白质和RNA乙酰转移酶的研究（Sas 2/4/5， Caf1和Nat10），N-末端蛋白乙酰转移酶（NAT）调节（NAT复合物和核糖体），以及 小分子乙酰转移酶（ACLY、ACSS 2、FATS2、ACCl）。目前没有质量 校园内的光度计或类似仪器。该仪器将位于一个专门的生物物理 Marmorstein实验室的设备室和Marmorstein博士的生物化学系 和生物物理致力于在整个过程中正确安装和维护质量光度计。 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.