A large range of debilitating medical conditions1 is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains2. 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 misfolding3. Here we use single-molecule Förster resonance energy transfer4,5 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 domains3. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent3. 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.
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This work was supported by the Wellcome Trust (grant number, 064417), the Swiss National Science Foundation (to B.S.) and the Swiss National Center of Competence in Research in Structural Biology (to B.S.). M.B.B. was supported by a UK Medical Research Council studentship. A.B. is supported by a Marie Curie Intra-European Fellowship. R.B.B. is supported by a Royal Society University Research Fellowship. J.C. is a Wellcome Trust Senior Research Fellow. We thank H. Hofmann, A. Soranno and A. Hoffmann for discussions and contributions to data analysis.
The authors declare no competing financial interests.
The file contains Supplementary Methods, Supplementary Table 1, Supplementary Figures 1-9 with legends and additional references. (PDF 8146 kb)
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