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Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-envelopedT = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.

Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.