States and transitions during forced unfolding of a single spectrin repeat.
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 alpha-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 force-extension 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.
European Molecular Biology, Cell Biology and Biophysics Programme, P. O. Box 10.2209, D-69012, Heidelberg, Germany. lenne@embl-heidelberg.de
P F
PF
Lenne
A J
AJ
Raae
S M
SM
Altmann
M
M
Saraste
J K
JK
Hörber
D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002645:Chickens; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D013049:Spectrin