Spectrin domains lose cooperativity in forced unfolding
Lucy G Randles 1, Ross W S Rounsevell 1 and Jane Clarke 2*
1 University of Cambridge
2 University Of Cambridge
* To whom correspondence should be addressed. E-mail: jc162{at}cam.ac.uk.
Submitted on July 19, 2006
Revised on September 10, 2006
Accepted on 10 October 2006
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Abstract |
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Spectrin is a multidomain cytoskeletal protein, the component 3-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies neighbouring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, non-helical linkers (all-beta immunoglobulin domains) were used to avoid non-native helical linkers forming. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behaviour observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behaviour allows multidomain proteins maintain their integrity under applied force.
Key Words:
AFM, Spectrin, cooperativity, domain: domain interactions, protein folding, titin
