Molecular Mechanisms, Pathways and Inhibition of Acetyl-Transfer Reactions

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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.