States and transitions during forced unfolding of a single spectrin repeat

Abstract

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 K K-helices that form a coiled-coil structure. Spectrin forms an oligomeric structure that is able to cross-link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, 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. The forceê xtension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states. ß

Influence of Lateral Association on Forced Unfolding of Antiparallel Spectrin Heterodimers

“…Lenne et al (10) found a similar length for a 4-mer of a single spectrin repeat flexibly linked in series. Data for actinin homodimer appear similar to that for the spectrin heterodimer, showing a bimodal distribution with a factor of ϳ2.0 between the mean for the major and minor peaks (data not shown).…”

“…Surprisingly, perhaps, recent single-molecule studies (1,9,10) show that individual spectrin repeats will unfold under modest tensile forces. This is consistent with such motifs contributing directly to cytoskeletal flexibility and strain softening (11).…”

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the αβ spectrin self-association binding site

“…2 Refinements and phasing of the model have been made as determination of the primary structure, 3,4 X-ray, nuclear magnetic resonance, and spectroscopic studies of spectrin have been completed. 28,[35][36][37][38][39][40][41][42][43] These studies predict that the first and third helices are parallel and the second helix is antiparallel. The amphipathic repeats are stabilized by hydrophobic interactions of each repeat at the interior core of each repeat.…”

The interplay between chemistry and mechanics in the transduction of a mechanical signal into a biochemical function

“…Those modules were found to unfold at forces in the range of 100-300 pN, at loading rates of the order of 10 -5 N/s. The alpha helix domains unfold instead, in the same conditions, at forces almost one order of magnitude smaller [73][74][75]. Also barnase, a beta-sheet rich protein without any putative mechanical role in vivo, seems to unfold at low forces in the same conditions [76].…”

“…In particular, the unfolding of the proximal Ig domains of titin may serve as a buffer to protect cardiac sarcomeres from being damaged at forces exceeding the physiological range [74].…”