Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.