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The need for multi-domain studies

Protein domains are the evolutionary, structural and functional units of proteins. Some of these domains exist in isolation, however ~ 70% are found in larger multi-domain proteins. The majority of the folding studies completed to date have been on individual domains. Neighbouring domains may be independent or there can be an effect on both the stability and the kinetic behaviour in multi-domain constructs. We aim to observe these changes and to see if there is any indication from the structure alone as to whether there will be folding dependence between domains.



Titin domains: Titin is a large multi-domain protein found in the sacromere which is responsible for muscle elasticity. The I-band contains a tandem array of immunoglobulin (Ig) like domains separated by linkers with little structure. The domain dependence has been studied by thermodynamics, kinetics and force measurements and there appears to be no effect on one domain by an adjacent domain. These domain are said to fold independently1.

Fibronectin domains: Fibronectin domains are highly abundant and are found in proteins with diverse functions. They also have an Ig-like fold. Two fibronectin domains from human fibronectin (FNfn9 and FNfn10) have been studied. A previous study indicated that FNfn9 relied on a neighbouring FNfn10 domain to have its full stability indicating a folding dependence between domains. However, we found that extending the FNfn9 domain by just 2 residues increased the stability of FNfn9 alone to that in a FNfn9-FNfn10 construct. This shows how important it is to define the correct domain boundaries2.

Spectrin domains: Spectrin repeat domains are three helix bundle coiled-coil domains involved in maintaining cell shape and elasticity. The spectrin repeats are not separated by linker with little structure, as in the Ig-like domains, but by an extended helix containing the C-helix of the N-terminal domain and the A-helix of the C-terminal domain. Although the spectrin domain can fold independently both the thermodynamic and kinetic stability are increased by neighbouring domains3-6.


Folding and misfolding of TI I27

The 27th Ig-like domain (in the I-band region) of the multidomain muscle protein Titin has been greatly investigated by the group. The structural core was determined by Φ-value analysis and this core is shared with other unrelated Ig-like domains7.

Unusual unfolding behaviour was observed for I27, characterised by downward curvature of the unfolding arm of its chevron plot. This was attributed to the switching of parallel pathway dominance with increasing [GdmCl]8.

Kinetics of the isolated wild-type domain were established by ensemble measurements and shown to be representative of the domain in multidomain constructs1. This representative quality was also demonstrated for the I29-32 domains but interestingly the kinetics of the 28thIg-like domain were shown to be situation sensitive.

The sequence similarity of neighbouring domains was demonstrated to be significant with regards to aggregation, with > 40% identity leading to greater aggregation9. This is a very important observation as it goes some way to explaining how multidomain proteins are adapted to prevent aggregation in vivo.

Single molecule FRET experiments showed that for repeats with > 40% sequence identity a misfolded species was formed in samples that had been unfolded and refolded previously. This species accounted for ~5% of the total population. This effect was not seen in constructs where the domains involved had very little identical sequence10.



  1. Scott, K. A., Steward, A., Fowler, S. B. & Clarke, J. (2002). J. Mol. Biol. 315, 819-829.
  2. Steward, A., Adhya, S. & Clarke, J. (2002). J. Mol. Biol. 318, 935-940.
  3. Batey, S., Randles, L. G., Steward, A. & Clarke, J. (2005). J. Mol. Biol. 349, 1045-1059.
  4. Batey, S., Scott, K. A. & Clarke, J. (2006). Biophys. J. 90, 2120-2130.
  5. Batey, S. & Clarke, J. (2006). Proc. Natl Acad. Sci. USA 103, 18113-18118.
  6. Randles, L. G., Rounsevell, R. W. S. & Clarke, J. (2007). Biophys. J. 92, 571-577.
  7. Fowler, S. B. & Clarke, J. (2001). Structure 9, 355-366.
  8. Wright, C. F., Lindorff-Larsen, K., Randles, L. G. & Clarke, J. (2003). Nat. Struct. Biol. 10, 658-662.
  9. Wright, C. F., Teichmann, S. A., Clarke, J. & Dobson, C. M. (2005). Nature 438, 878-881.
  10. Borgia, M. B., Borgia, A., Best, R. B., Steward, A. Nettels, D., Wunderlich, B., Schuler, B. & Clarke, J. (2011). Nature 474, 662-665.