Doctor of Philosophy
School of Chemistry
Blayney, Michelle J., Mass spectrometric investigations of biomolecular machines, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3316
Nanoelectrospray ionisation (nanoESI) coupled with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has developed to the point that noncovalent complexes of biomolecules can be analysed. In this work, current MS technology was used to analyse protein assemblies of tobacco rubisco and rubisco activase and their interactions with nucleotides and sugar phosphates. The first nanoESI mass spectra of rubisco and rubisco activase are presented. Rubisco activase was determined to have a higher relative binding affinity for ADP over AMP-PNP (and by inference, ATP) and the addition of ATP-Mg revealed a possible second nucleotide binding site. This is the first clear evidence of a second nucleotide binding site on rubisco activase and binding at the first site potentiated binding at the second site. Further, an interaction between rubisco activase and RuBP, but not CABP was observed. While this observation should be treated with caution, the binding of RuBP to rubisco activase suggests direct involvement of rubisco activase in removing RuBP from the inactive rubisco-RuBP complex. The inability to observe a rubisco activase-CABP complex suggests a specific interaction of rubisco activase with RuBP. The addition of rubisco activase to the inactive rubisco-RuBP complex led to removal of RuBP from rubisco, though no direct complex between rubisco and rubisco activase was observed by ESI-MS. In addition, the oligomeric assembly of an archaeal rubisco from Methanococcoides burtonii was investigated. In the presence of substrates, RuBP or CABP, or divalent metal cofactors, M. burtonii rubisco assembled from dimers into decamers. The nanoESI mass spectra confirmed results observed using native polyacrylamide gel electrophoresis. These results suggest a re-examination of the classification of the rubisco superfamily since the M. burtonii rubisco has catalytic and structural similarities with other archaeal rubiscos, but closer sequence homology to bacterial type II rubiscos. NanoESI-MS was also used to investigate protein subassemblies of the DNA polymerase III of the E. coli replisome. This molecular machine is responsible for the replication of chromosomal DNA prior to cell division. The interaction between the 2 sliding clamp and the polymerase subunit was the focus of this investigation, in particular, characterisation of the two 2-binding sites on the subunit, as well as preliminary work to determine conditions toward the assembly of larger subassemblies of the replisome. Complexes of the core () subassembly with the 2 sliding clamp, and core with the truncated subunit, c16, were observed. Determination of the conditions for observation of these complexes has set the scene for more detailed analysis of the order of assembly of the replication machinery and for an understanding of the protein binding partners involved in key interactions. Based on the observations made in this work, evidence has recently been obtained using nanoESI-MS to show that the proofreading subunit interacts directly with the 2 sliding clamp. Finally, travelling wave ion mobility mass spectrometry (TWIMS) is a relatively new type of IMS. In IMS, ions are separated not only on the basis of mass/charge, but also by their size and shape. Since this a newly commercially available technology, it was of interest to determine the resolving power of the Synapt™ HDMS™ instrument for protein conformers. A mixture of 2 and transferrin, proteins of similar mass but different shape, was resolved using TWIMS. TWIMS was also used to confirm that changes in charge state distribution can correspond to protein unfolding, where the unfolded form of DnaB-N was separated from the folded conformation. Differences in collisional cross section were determined from TWIMS data of complexes of calmodulin in positive or negative ion mode. In these studies, mass spectrometry provided information that is not readily available from other techniques.