Pushing the limits of Structural Mass Spectrometry to Characterize the Viral Packaging Motor
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Assessing protein structure is a difficult undertaking, yet is often required for a complete mechanistic understand of how a protein functions. The traditional techniques used to assess protein structure are X-ray crystallography and NMR, which both yield high-resolution data. Despite the success of these techniques, NMR and crystallography are not high-throughput techniques, have size limitations, require protein crystals, and/or require high concentrations of protein purified to homogeneity. Alternative techniques are required to probe the structures of proteins that are difficult to purify and rapidly characterize protein structure. Toward this end, structural mass spectrometry techniques provide a powerful alternative due to high sensitivity, versatility, and potential for fairly detailed structural information. All of the work described in this thesis concerns structural MS in some capacity. A major focus of this work is the bacteriophage lambda packaging motor, called terminase. Terminase is a complex, multi-subunit viral enzyme that is one of the most powerful molecular motors known. This enzyme packages a double stranded DNA genome into empty viral capsids at a rate of 600 base pairs per second, which generates over 20 atmospheres of internal capsid pressure. The packaging of the viral genome by a terminase enzyme is crucial for producing new infectious virus for many dsDNA viruses such as adenovirus, herpesvirus, and several bacteriophages. In the first chapter, new structural MS technologies are developed and discussed. A new ionization technique, called surface acoustic wave nebulization (SAWN), was coupled with digital microfluidics (DMF) to show a novel workflow that could lead to a lab-on-a-chip type workflow for hydrogen-deuterium exchange (HDX). In DMF-SAWN-HDX, a 5 µl peptide sample was successful merged with deuterated water, transferred to the SAWN chip for nebulization and analyszed by MS. Additionally, SAWN-HDX alone was shown to be effective at measuring HDX of ubiquitin, and the SAWN-HDX data of ubiquitin were corroborated with NMR HDX ubiquitin data. I then demonstrate the utility of SAWN-MS for a novel application: the structural characterization of noncovalent interactions. Comparisons of SAWN with ESI showed several potential advantages of SAWN over ESI. SAWN-MS analysis of myoglobin resulted in less heme dissociation when analyzing myoglobin in comparison to ESI, and SAWN had less evidence of nonspecific noncovalent interactions. SAWN of the TerS-A55 deletion mutant containing the DNA binding domain of the small terminase subunit led to little to no dissociation of the dimer, while ESI disrupted significantly more of noncovalent interactions. SAWN-HDX studies of TerS-A55 were found to exhibit solvent protection at the dimer interface and SAWN-HDX demonstrated a clear decrease in deuterium uptake in the presence of DNA. Finally, I discuss the development and application of cross-linking MS (XLMS) to study terminase. I validated a new means of analyzing XLMS data, which resulted in the identification of more cross-links in comparison to other methods. XLMS data was further validated with cross-linking data from proteins with known structures, and this data showed that cross-links accurately reflect the native solution structure. XLMS was then applied to lambda terminase, a protein with unknown structure, in combination with homology modeling. These results provide valuable structural insight into the structure of TerS and the packaging mechanism. Overall, the work described here advances structural MS and our understanding of lambda terminase. Novel structural MS technologies and workflows are described, which provide a future means to streamline workflows, improve analysis, yield higher quality data, increase the accessibility of certain structural MS techniques, and provide several avenues for continued future development. The last chapter provides valuable structural insight into the assembly of the TerS dimer and the structural plasticity within the TerL subunit, which has important mechanistic implications. This work also more broadly presents a detailed means for how to approach structural characterization of proteins that are difficult to purify and analyze by traditional structural methods.
- Medicinal chemistry