17th International Mass Spectrometry Conference :: Prague, 2006
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|Speaker:||Alison E. Ashcroft|
|Presentation date:||Tue, Aug 29, 2006|
|Presentation time:||12:10 – 12:30|
Alison E. Ashcroft1, Ottar Rolfsson1, Simona Francese1, Gary S. Thompson1, Nicola J. Stonehouse1, Peter G. Stockley11 University of Leeds, Leeds, United Kingdom
Correspondence address: Alison E. Ashcroft, University of Leeds, Faculty of Biological Sciences, Astbury Centre for Structural Molecular, Mount Preston Street, Leeds, West Yorkshire, LS2 9JT United Kingdom.
Web site: http://www.astbury.leeds.ac.uk
Keywords: Complex, Non-Covalent; Electrospray Ionization (ESI); Protein; Protein Conformation.
Novel aspect: Virus assembly intermediates detected, characterised and shown to be on pathway by ESI-MS for the first time.
The precise mechanism of capsid formation for simple viruses remains elusive. These details are much sought after, not only for virology purposes but also because viral capsids have a number of potential bio-nanotechnology applications.
RNA phages are models for many viral properties, and hence can provide much useful information in this area. One such example is the bacteriophage MS2, in which it is known that reassembly of a T=3 shell can be initiated by a sequence-specific RNA-protein interaction. The minimum RNA sequence that will catalyse this reaction is encompassed within a 19 nucleotide stem-loop (TR), whilst the quasi-equivalent conformational changes required to form the capsid occur in a single loop, the FG-loop, of 180 identical coat protein subunits packaged as non-covalently bound dimers (CP2).
Using ESI-MS we have monitored the assembly of MS2 capsids from the CP2 and TR reactants. In order to preserve the non-covalently bound protein-RNA complexes, the mass spectrometer used for these studies was a customised LCT Premier which has an extended m/z range (m/z 60,000) and has been adapted for collisional cooling. Sample introduction and ionisation were achieved by the use of a NanoMate autosampling device with nano-electrospray ionisation.
Addition of TR to CP2 results in the formation of an initial [CP2+TR] complex, accompanied by small amounts of higher order species. Addition of further CP2 dramatically increases the amounts of these species, which then disappear over time concomitantly with capsid production, the latter verified by electron microscopy. This series of events supports the supposition that the viral control assembly mechanism maintains the CP2 in an assembly-incompetent state until interaction with the TR ligand is possible.
Initiation of the assembly mechanism with 15N-labelled CP2 and TR, followed by the addition of unlabelled CP2, indicates clearly that the higher order species are all competent for further assembly and thus are assembly intermediates rather than extraneous species or mass spectral artefacts. The results also have implications for the assembly pathway to the final T=3 shell from the essential CP2 building block.
Data from assembly mechanisms using longer genomic RNA species and coat protein variants will also be presented, and the role of these species in virus assembly mechanisms discussed.