Analysing macromolecular structures with native mass spectrometry : insights to virus structure and assembly

In the past years, native mass spectrometry (MS) has gained attention in research and also industry. The technique can provide information on mass, topology, binding affinity or stability and dynamics of macromolecular structures. With ion mobility (IM) even conformational changes can be monitored. With this technique, viral protein complexes involved in capsid formation were analysed. These nanocontainers enclose the genome in viruses. Even though studied for decades, many questions remain, for example, whether the capsid assembly proceeds to completion. For Hepatitis B virus (HBV), charge state distributions could be resolved for the two capsid species. The assigned masses of ~ 3 and 4 MDa (standard deviation below 0.1%) were in agreement with completely assembled capsids consisting of 90 or 120 dimers as also confirmed by collision induced dissociation (CID). The two HBV capsids have similar internal energies in the gas phase enabling a direct comparison of their stability in CID. Interestingly, the smaller assembly exhibited a higher resistance to CID in agreement with previous mutagenesis studies. However, the mechanical properties of the two geometries were similar in atomic force microscopy (AFM) enforcing that the relation between gas phase and in solution stability is not trivial. Furthermore, theoretical models predicted exchange of dimers between capsids and free ones in solution. I monitored the dynamic incorporation of isotopically labelled dimers into preassembled HBV capsids using CID on selected species. A slow exchange occurred over months exclusively at low temperatures and independent of solution pH, but only in the smaller capsids. IMMS provides structural information at low resolution and is often combined with computational modelling. The question arises to which degree structures are preserved in the gas phase. For the large viral assemblies, shape determination in IMMS was possible with some adjustments. Remarkably, the HBV capsids exhibit a vacuum shape consistent with the hollow spheres observed in electron microscopy or AFM and with dimensions modelled for the two species. Of special interest to vaccine and drug development are viral assembly and disassembly pathways. We found that the norovirus, causing viral gastroenteritis, shows a complex albeit reversible dissociation and reassociation behaviour dependent on pH and ionic strength. The detected oligomers including a 60-mer, an 80-mer and the 180-mer capsid likely interconverted via dimers. Surprisingly, the 60-mer and 80-mer also resembled hollow capsid-like structures in IMMS and AFM. The extraordinarily high sensitivity in IMMS enabled studies on norovirus and also HBV oligomers, which are inaccessible by common techniques in structural biology. Comparison of data on the viral intermediates and globular protein complexes as well as modelled results indicated that the intermediates have extended structures as in assembled capsids. Further analysis provided insights into the priming of and a common pathway for assembly in both viruses. In this thesis, I have shown that the structure, stability and dynamics of protein complexes even in the megadalton range can be analysed by native MS in combination with IM and modelling. Next to the implications for structural biology or virology, these methods hold high potential in nanotechnology