Confirmation of intersubunit connectivity and topology of designed protein complexes by native MS

Abstract

<p id="d7435716e270">Recent advancements in computational protein design have enabled the generation of proteins and protein complexes that are not present in nature. A current major limitation of the research is in the subsequent structural characterization. The commonly used techniques—X-ray crystallography, NMR spectroscopy, and cryo-EM—are time- and resource-consuming and heavily rely on sample quantity and quality. These limitations are particularly problematic for protein complexes. In this work, we show the applicability of native MS ion mobility coupled with surface-induced dissociation as a structural biology screening tool for designed proteins. We show that information about stoichiometry, intersubunit connectivity, and complex topology is rapidly gained by this SID-IM-MS methodology. </p><p class="first" id="d7435716e273">Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein–protein interactions to generate specific protein assemblies. In this work, we designed protein–protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches. </p>