Figure 6.

Direct single molecule measurements of fast and intermediate dissociation time constants. (A) Representative data for measuring protein dissociation at constant force. Overstretched dsDNA (solid black) is incubated in 2 nM 111p for 360 s. The returning 111p–DNA complex after incubation (open blue circles) is stopped and maintained at a constant force of 43 pN via a force feedback loop for 360 s. (B) The change in extension with time during the constant force feedback loop (green circles) is fit to a double exponential function of time (solid black). Two time constants τ_int and τ_fast represent the characteristic dissociation time constants of the ssDNA-bound 111p populations exhibiting respectively intermediate and fast dissociation kinetics. (C) Variation of the time constants with stopped force for ORF1p variants. Intermediate (circles) and fast (triangles) dissociation time constants are measured as a function of a stopped force (F_st). Green, blue and red data points correspond to: 111p, 555p and 151p respectively. Overall variant averages are fit to an exponential function of force, τ(F) = τ₀e^F/Ω, using the minimization of χ² method (dashed gray lines), where Ω is a factor that describes the scale of time constant variation with force. Corresponding fits for τ_f and τ_i yield τ_0,fast = 2.7 ± 0.4 s, Ω_fast = 28 ± 1 pN and τ_0,int = 57 ± 4 s, Ω_int = 75 ± 10 pN respectively. Error bars are standard errors for at least three measurements (also see Supplementary Figure S4 and Table S3).