Multiple modes of AFM reveal distinct mechanical properties for dystrophin and utrophin not manifest by small fragments

Figures

• 

  Fig. 1.. Single molecule force spectroscopy of dystrophin and utrophin. (A) Domain structure of Dys, DysN-R10, Dp260, and Utr. Ovals, spectrin-like repeats (SLRs); diamonds, hinge domains; NT, N terminus; CT, C terminus; CR, cysteine-rich domain; ABD1 & 2, actin-binding domains; and DgBD, dystroglycan binding domain. (B) Coomassie blue-stained SDS polyacrylamide gel with purified proteins. (C) Illustration of the SMFS experiment.

• 

  Fig. 2.. SMFS using two operational modes of AFM, constant speed and constant force. (A) A representative force-extension curve of molecule Dys obtained in constant speed mode. (B) Unfolding force histograms with fitted kernel density estimation from one biological replicate of molecule Dys at 1,000 nm/s. (C) A representative curve of molecule Dys from constant force mode, with the extension-time curve in the Top panel and the force-time curve in the Bottom panel. (D) Lifetime histograms with fitted kernel density estimation from one biological replicate of molecule Dys at 70 pN. Unfolding events are highlighted with blue circles in both (A and C).

• 

  Fig. 3.. Statistical summary of N=3 biological replicates for (A) constant speed and (B) constant force experiments. Each column presents results for different molecules, namely Dys, DysN-R10, Dp260, and Utr from Left to Right. The data from three biological repeats are visualized in purple, pink, and yellow. Unfolding force distributions from constant speed experiments and lifetime distributions from constant force experiments are depicted using violin plots. The violin plots illustrate data distributions using kernel density estimation as black lines on each side, with the width of each curve indicating the relative frequency of data points. Meanwhile, black stars signify the positions of values with the highest probability, visualized by the widest section of the violin plot, and red triangles indicate the lifetime averages, calculated from identical forces and replicates.

• 

  Fig. 4.. Protein mechanical behavior as a function of unfolding event count (the number of domains unfolded). (A) Most probable forces from constant speed experiments. (B) Mean dwell lifetime, the time interval between consecutive unfolding events, derived from constant force experiments. Error bars represent 95% CIs, calculated using bootstrapping.

• 

  Fig. 5.. Transition rates and energy landscapes of four molecules, Dys, DysN-R10, Dp260, and Utr. (A) koff-vs-F curves showing mean values (lines) and SDs (error bars). (B) Cusp-like (ν=1/2) energy landscapes and (C) linear cubic (ν=2/3) energy landscapes, with mean values (lines) and SDs (shaded area). Mean and SDs are computed across different operational modes and biological replicates.