Low force cooperativity in the extensible unfolding of cytoskeletal proteins

Spectrin family proteins are flexible, cytoskeletal linkage components with common three-helix repeats but diverse self-association states. Red cell a and b- spectrins are known to associate along their lengths. The effect of such lateral interactions on chain flexibility, especially extensible unfolding, has been probed by AFM. In large ensemble experiments on two to four repeat constructs of a & b spectrin, either as monomers or as ab-dimers, sawtooth patterns of extension reveal domain unfolding that stochastically extends repeats 6-7 fold greater in length than native conformations. Distributions of unfolding lengths or periods appear bimodal: the major unfolding peak corresponds to a single repeat and the minor unfolding peak at twice the length corresponds to a tandem series of repeats, consistent with a contiguous helix between repeats in recent crystal structures. The force f for either event is the same, but a lower probability for the tandem events appears consistent with barrier to unfolding pairs of repeats. With ab-dimers, unfolding requires 1.6-fold higher f, whereas parallel unfolding of domains in two unassociated monomers requires 2.0-fold higher f. Cooperativity in forced unfolding of spectrin is thus implicated by (i) a common f for single and tandem repeats, and (ii) easier unfolding of associated versus unassociated chains.