We have characterized the force-dependent kinetics of unfolding and refolding for spectrin, an important cytoskeletal protein. This was accomplished by using forward and reverse Dynamic Force Spectroscopy, which employs both ascending and descending ramps of force.

  1. Identification of unfolding/refolding transitions under mechanical force

  2. -Transitions identified by abrupt tether length changes

  3. -Force-extension allows identification of molecular state

Identifying transitions: Bead height vs. time for three cycles (left) and corresponding force-extension (right) with arrows labeling transitions. Superposition of 30 cycles (right inset) fit with a multi-branch worm-like chain (persistence length = 1.0(1) nm, contour length = 98+m·32(1) nm, where m is the number of unfolded domains.

relevent group publications

E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, “A new approach to analysis of single molecule force measurements,” in Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). [link]

K. Halvorsen, “Probing weak single-molecule interactions: development and demonstration of a new instrument,” Ph. D Thesis (advisor: E. Evans), Department of Biomedical Engineering, Boston University, USA (2007). [pdf]

W.P. Wong, “Exploring single-molecule interactions through 3D optical trapping and tracking: from thermal noise to protein refolding,” Ph. D Thesis (advisors: D.R. Nelson and E. Evans), Department of Physics, Harvard University, USA (2006). [pdf]

  1. Unfolding: Forward DFS

Unfolding/Refolding data: the unfolding/refolding transition force was measured for different increasing/decreasing force loading rates. Figures shows histograms of spectrin transition forces, and the most-likely/mean transition force vs. log(|loading rate|) with the model superimposed.

  1. Refolding: Reverse DFS

  1. Unfolding-refolding “oscillations” and observation of a metastable state

  2. -Bistable unfolding/refolding “oscillations” observed during slow decreases in force

  3. -Metastable state is much less kinetically stable than the fully folded state, as quantified with MLE

Metastable state: Bead height vs. time with metastable oscillation (left) and lifetime data fit with a decaying exponential (right).