Single-molecule fluorescence reveals sequence.pdf

LETTER doi:10.1038/nature10099 Single-molecule fluorescence reveals sequence- specific misfolding in multidomain proteins Madeleine B. Borgia1,2, Alessandro Borgia2, Robert B. Best1, Annette Steward1, Daniel Nettels2, Bengt Wunderlich2, Benjamin Schuler2 Jane Clarke1 A large range of debilitatingmedical conditions1 is linked to protein misfolding, whichmay 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 thatmaintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding3. Here we use single-molecule Fo?rster resonance energy transfer4,5 to detect and quantify raremisfoldingevents in tandemimmunoglobulindomains fromthe I bandof titinundernative conditions.About 5.5per centof 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 neigh- bouring domains than between non-adjacent domains3. In particu- lar, the sequence identity of neighbouringdomainshas been found to be preferentially below 40 per cent3. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence iden- tity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations pre- dict the formationofdomain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species.We infer that the interactions underlyingmisfolding are very specific and result in a sequence-specific domain-swapping mech- anism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multido