From structure to function : Protein assemblies dissected by mass spectrometry

This thesis demonstrates some of the possibilities mass spectrometry can provide to gain new insight into structure and function of protein complexes. While technologies in native mass spectrometry are still under development, it already allows research on complete proteins and protein complexes up to a seemingly unlimited size. This would not have been possible without the technical developments in all related fields, for example ionization, instrumentation and sample preparation and handling. An example for further instrumentation development is given in chapter two of this thesis. Here we attempted to improve one of the instruments frequently used in native mass spectrometry, the Q ToF, by modifying the pressures, nature of collision gas and orifi ces of the collision cell. These changes on the Q ToF allowed us to obtain the results that are described in chapter 2 to 4 of this thesis. Nowadays mass spectrometry aims to bridge between data of high throughput proteomic screens of protein networks in the cell and high resolution structural data obtained by methods like x ray, cryo EM and NMR. The most direct result obtained by native mass spectrometry for protein complexes is often the stoichiometry. While with homogeneous complexes this is visible at fi rst sight most of the times, heterogeneous complexes often need to be dissociated by tandem mass spectrometry. In chapter 3 we determined the stoichiometry of the purifi ed portal ring and the assembly of the portal ring with the fi rst tail accessory factor gp4 of bacteriophage P22. Besides determining the stoichiometry we were also able to make conclusions about the binding behavior and structural changes that occur upon the assembly. Charge state distributions generated in the ESI process arouse the suspicion that the portal might undergo a major conformational change upon binding of gp4. A new technique called ion mobility mass spectrometry confi rmed this structural change. These results gave new insights into the maturation process of P22. Several interactome studies have shown that it is possible to establish interaction networks of endogenously expressed proteins in yeast by affi nity pulldowns combined with mass spectrometry. While these studies have shown that proteins almost always work in interaction with other proteins they fail to give information about the structure and stoichiometry of the proteins. Native mass spectrometry can provide new and complementary information here. We have analyzed the architecture of the multisubunit RNA polymerases Pol II and Pol III. The measurements presented in chapter 4 resulted in insights into the subunit architecture of Pol II and III. We here compared the results gained from Pol II, where detailed structural properties are known, with Pol III. The data demonstrates that each of the 17 subunits of Pol III is present as a single copy. Our analysis provided new information about the heterodimeric subcomplexes in Pol III. The obtained model for the Pol III subunit architecture might guide future biochemical and structural studies of this central cellular enzyme