Characterisation of Noncovalently bound Protein-RNA Complexes and their role in Virus Capsid Assembly Mechanisms using ESI-MS

摘要

A major unresolved problem in structural virology is to understand the detailed molecular mechanism that gives rise to the assembly of capsids of the correct size and symmetry. Capsid assembly is spontaneous and usually very rapid, hence traditionally it has been technically difficult to isolate and characterise intermediates beyond the initiation complexes on the pathway to the final products. The RNA bacteriophage MS2 is an ideal model for investigation as it is known that reassembly of its capsid, in which 180 identical coat protein (CP) subunits are packaged as noncovalently bound dimers (CP_(2)) in a T(velence)3 shell, can be initiated by a sequence-specific noncovalent protein-RNA interaction (Figure 1). We have monitored the entire capsid assembly course of MS2 and have identified a key, time-dependent intermediate using nanoESI-MS. Using isotope pulse-chase experiments we have shown that these higher order species are on-pathway to capsid formation. The data suggest that conformational changes in the coat protein dimer affect the assembly mechanism, and we have used ESI-travelling wave ion mobility spectrometry-MS (ESI-TWIMS-MS) to study the conformational properties of the coat protein.