Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry.

We have combined ion mobility spectrometry-mass spectrometry with tandem mass spectrometry to characterise large, non-covalently bound macromolecular complexes in terms of mass, shape (cross-sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision-induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring-like three-dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on-pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross-sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X-ray structure of the coat protein building blocks. Comparing the cross-sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3-fold and the 5-fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti-viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non-covalently bound macromolecular complexes and their assembly pathways.

RNA-induced conformational changes in a viral coat protein studied by hydrogen/deuterium exchange mass spectrometry.

A detailed knowledge of the capsid assembly pathways of viruses from their coat protein building blocks is required to devise novel therapeutic strategies to inhibit such assembly. In the quest for understanding how assembly of single-stranded RNA viruses is achieved at the molecular level, HDX-MS has been used to locate regions of a coat protein dimer that exhibit conformational/dynamical changes, and hence changes in their HDX kinetics, upon binding to a genomic RNA stem-loop known to trigger assembly initiation. The HDX-MS data highlight specific areas within the coat protein dimer that alter their exchange kinetics in the presence of the RNA. These include the known RNA-binding sites, ß-strands E and G, which have a lower susceptibility to HDX when ligand-bound, as may have been expected. In contrast, several exposed regions are unaffected by ligand binding. Significantly in this example, the loop between ß-strands F and G exhibits reduced HDX propensity when the RNA is bound, consistent with previous inferences from NMR and normal mode analysis that suggested a local conformational change at this loop induced by dynamic allostery. These results demonstrate the potential utility of HDX to probe conformational and dynamical changes within non-covalently bound protein-ligand complexes which are of widespread importance in many biomolecular systems.

Insights into virus capsid assembly from non-covalent mass spectrometry.

The assembly of viral proteins into a range of macromolecular complexes of strictly defined architecture is one of Nature's wonders. Unraveling the details of these complex structures and the associated self-assembly pathways that lead to their efficient and precise construction will play an important role in the development of anti-viral therapeutics. It will also be important in bio-nanotechnology where there is a plethora of applications for such well-defined macromolecular complexes, including cell-specific drug delivery and as substrates for the formation of novel materials with unique electrical and magnetic properties. Mass spectrometry has the ability not only to measure masses accurately but also to provide vital details regarding the composition and stoichiometry of intact, non-covalently bound macromolecular complexes under near-physiological conditions. It is thus ideal for exploring the assembly and function of viruses. Over the past decade or so, significant advances have been made in this field, and these advances are summarized in this review, which covers the literature up to the end of 2007.

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9.

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.

The impact of viral RNA on assembly pathway selection.

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T=3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T=3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.

Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid.

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP(2)) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T=3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qbeta known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T=3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP(2). We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5' or 3' end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T=3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T=3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.