Mass spectrometry as a tool for structural biology: crosslinking‐MS and native‐MS methods to study the RNA Pol II ‐ Mediator holoenzyme (778.6)

Abstract

The Mediator complex plays a central role in transcriptional regulation by relaying gene specific regulatory signals to the general transcriptional machinery. The yeast holoenzyme between mediator and RNA pol II consists of 33 subunits totaling over 1.5 MDa in size. The large size precludes atomic resolution crystallography of the entire assembly. Hence hybrid methods of structure determination are necessary, integrating various techniques including: crystallography of stable subassemblies, cryoEM of the intact complex, and mass spectrometry (MS). Mass spectrometry based structural techniques including chemical crosslinking and native‐MS provide spatial restraints that guide and constrain the modeling process. Crosslinking measurements map interacting protein‐protein surfaces through the identication of amino acid residues that have been covalently modified by heterobifunctional reagents. Native MS permits the determination of stably associated assemblies of proteins in the gas phase. We focus on the development of these new MS based technologies including the design of robust bioinformatics strategies, enrichment methods, and reagents for optimizing mass spectrometric analysis of crosslinked peptides, as well as deconvolution algorithms and sample preparation for native MS analysis. We discuss the application of these methods to determining the structure of the Mediator holoenzyme.Grant Funding Source: Supported by: NIH P41GM103481