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

A large range of debilitating medical conditions(1) are linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains(2). With 75% of the eukaryotic proteome consisting of multidomain proteins, how is inter-domain misfolding 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 (FRET) experiments(4,5) to detect and quantify rare misfolding events in tandem Ig domains from the I-band of titin under native conditions. About 5.5% of molecules with identical domains misfold during refolding in vitro and form a surprisingly stable state with an unfolding half time of several days. Tandem arrays of immunoglobulin-like (Ig-like) domains in humans exhibit 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%(3). Interestingly we observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24%), whereas misfolding occurs between domains which are 42% identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures, which 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 appears to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.