Matthias Rief, Ingo Schwaiger, Hendrik Dietz, Thomas Bornschloegl
Lehrstuhl für Biophysik E22 der TU München, James-Franck-Str., 85748 Garching
The structural stability of cytoskeletal proteins and molecular motors are important for their function in vivo. However, this information has become accessible only recently through the invention of single molecule techniques. We have used AFM based force spectroscopy to investigate the mechanical response of the coiled-coil domains of myosin II and the actin cross-linking protein Ddfilamin.
We find that the myosin coiled-coil is a highly elastic protein structure that undergoes an unfolding/refolding transition at 25 pN. Unlike all other proteins investigated so far this transition occurs in equilibrium. These measurements show that a coiled-coil is able to produce forces during folding. Ddfilamin is an actin crosslinking protein from dictyostelium discoideum. Using single molecule unfolding experiments we show that one of the immunoglobulin domains of this protein unfolds at low forces via a stable intermediate. We have used amino-acid inserts into the loops of this domain to map the structure of this intermediate. We show evidence that the intermediate is also populated during folding of this domain which increases the refolding rates drastically. Low unfolding forces together with fast refolding kinetics suggest an in-vivo role for this domain as a reversibly extensible element under mechanical strain.
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