Advances in methodology for the analysis of DNA interacting proteins by native ion mobility mass spectrometry

  • Annika Kotter ((PhD) Student)
  • Frank Sobott (Promotor)
  • Filip Lemiere (Jury)
  • Sylvia Dewilde (Jury)
  • Remy Loris (Jury)
  • Bart Devreese (Jury)
  • Anne-Marie Lambeir (Jury)
  • Mark Dillingham (Jury)

Student thesis: Doctoral Thesis

Abstract

Many of the functions of nucleic acids (DNA, RNA) depend on their precise 3D structure, and how they interact with proteins. If we want to understand the molecular basis of diseases or infection, we have to understand those interactions during the cell cycle and protein expression. The proteins that interact with nucleic acids typically play important roles in the structure and activity of the genome. In this thesis I show that native ion mobility mass spectrometry (IM-MS) is a very useful method to study these proteins and their complexes with DNA. This includes analysis of the structure and composition of key complexes as well as their mode of operation. Native IM-MS allow us to get information about the mass, charge and also about the size and shape of the molecular complexes. This work also includes the development of novel methods for native MS of proteins and protein/nucleic acid complexes, and the use of modeling approaches to interpret the data and propose global structures of the assemblies.

Using a wide range of IM-MS related methods, I have optimized the use of this technique for DNA-interacting proteins and protein-DNA complexes. I found that recently described allosteric inhibitors have a different effect on the hepatitis C viral protein NS3/4A than protease inhibitors binding to the substrate binding site. The techniques I applied to this class of enzymes have, in conjunction with traditional methods such as ITC and H/D exchange, great potential for the rapid analysis of structural effects of drug candidates.

For the SMC proteins, I determined the stoichiometry of the complexes and showed previously unknown dimerization of the functional SMC complexes. Not only the SMC proteins, which are often assumed to be rod-shaped, but also their accessory proteins and other interaction partners exhibit structural flexibility in my experiments. For two different bacterial SMC systems, I showed differences but also similarities of their stoichiometries and mode of action. I explored the conformational landscape of the SMC dimers and mutants using different types of ion mobility analysis in conjunction with extensive computational modeling, as well as other MS-based methods (crETD, CIU) to assess structural models for the observed conformations in IM-MS and to show that they are not measurement artifacts. Finally, I determined the stoichiometry of Type III restriction enzymes and showed that one copy of the protein complex binds to one recognition site on DNA, without changing its stoichiometry. With addition of ATP, DNA is cleaved and the complex slides off the DNA, without evidence for large conformational changes.
Date of Award21 Mar 2016
Original languageEnglish
Awarding Institution
  • University of Antwerp

Keywords

  • Structural Biology
  • Protein-DNA interactions
  • Mass spectrometry

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