Measuring mechanical properties of protein structures with local probe methods

J.K. Heinrich Hoerber

EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so-called spectrin repeats. Each repeat contains three antiparallel a-helices that form a coiled-coil structure. The oligomeric spectrin structure is able to cross-link actin filaments. In red blood cells and in the hair cells of the inner ear such a spectrin/actin meshwork is thought to be responsible for special elastic properties of these cells. In order to determine mechanical properties of spectrin repeats, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. Precise force spectroscopy measurements at the single molecule level with the double detection AFM reveal states and transitions during the mechanical unfolding of spectrin and show that even single engineered cysteine bonds can be detected. The disulphide bond shortens the unfolding elongation, while they do not change the proteinｹs mechanical stability. After chemical dissociation of this bond, single spectrin domains recover their natural mechanical topology. The results demonstrate that the AFM can be used for the analysis of transition probabilities between different conformational states and that it is even sensitive enough to locate point mutations in individual protein domains by the resulting characteristic forced unfolding signature.