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Most proteins fulfil their function as part of large protein complexes. Surprisingly, little is known about the pathways and regulation of protein assembly. Several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virion and thus resemble ideal model systems for studying protein complex formation. Even for these systems, the mechanism for self-assembly is still poorly understood, although it is generally thought that smaller oligomeric structures form key intermediates. This assembly nucleus and larger viral assembly intermediates are typically low abundant and difficult to monitor. Here, we characterised small oligomers of Hepatitis B virus (HBV) and norovirus under equilibrium conditions using native ion mobility mass spectrometry. This data in conjunction with computational modelling enabled us to elucidate structural features of these oligomers. Instead of more globular shapes, the intermediates exhibit sheet-like structures suggesting that they are assembly competent. We propose pathways for the formation of both capsids. Although most proteins fulfil their role as part of large protein complexes, little is known about the pathways of complex assembly. Here, ion mobility–mass spectrometry is used to monitor and structurally characterize the assembly intermediates of viral protein shells, called capsids, of two major human pathogens, norovirus and hepatitis B virus.

Nature Chemistry 3, 126–132 (2011); published online 19 December 2010; corrected after print 23 July 2014. In the version of this Article originally published, the trace for cp4 was missing from Fig. 3a; it should have appeared as shown below. This has now been corrected in the online versions of the Article.

Using a capillary electrophoresis-based method, single enzyme molecule assays were performed on E. coli beta-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the enzyme. These samples were found to have substantially different distributions of single molecule activities. For the second set of samples, beta-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced enzyme had a 25% higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, beta-galactosidase was induced at 26 and 42 degrees C. The enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single molecule activities.