Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

When protein folding goes awry Three in four human proteins have multiple domains, and the tendency of these proteins to aggregate increases with amino-acid sequence similarity. Single-molecule biophysics experiments now show that the misfolding is caused by strand-swapping between adjacent domains, and that diversification of sequence between neighbouring domains lowers the probability of misfolding. The work also indicates that such misfolding events are under kinetic rather than thermodynamic control. Because domain-swapped protein species have been implicated in various misfolding diseases, prevention of inter-domain misfolding may be protective against aggregation disorders. A large range of debilitating medical conditions 1 is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains 2 . Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding 3 . Here we use single-molecule Förster resonance energy transfer 4 , 5 to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains 3 . In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent 3 . We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.