(A) Distributions of MIDAS-Rsa4 bond lifetimes (6 pN total force, 55 nt bridge construct) with either MIDAS-WT (n = 57 events from 14 molecules) or MIDAS-Y4666R (n = 29 events from 17 molecules). Survival probability is defined as one minus the empirical cumulative density function. For all distributions, the final data point was moved from y = 0 to y = 0.01 to enable semilog plotting. Black lines show fits to a single exponential. (B) Distributions of MIDAS-Rsa4 bond lifetimes on the 55 nt bridge construct at 4 pN (n = 26 events from eight molecules), 6 pN (n = 57 events from 14 molecules), and 12 pN (n = 29 events from five molecules) total applied force. (C) Distributions of MIDAS-Rsa4 bond lifetimes on the 70 nt bridge construct at 4 pN (n = 33 events from 11 molecules), 6 pN (n = 27 events from seven molecules), and 12 pN (n = 11 events from five molecules) total applied force. (D) The average bond lifetime of MIDAS-Rsa4 binding as a function of total applied force. Data shown as mean ± standard error of the mean (SEM; n = 26–57 events from 38 total molecules for the 55 nt bridge and n = 11–33 events from 28 total molecules; note that events at more than one force could be collected on a given molecule), with dotted lines to guide the eye. (E) Mechanical circuit model describing the force jump assay. When the proteins (of inextensible length Lprot) are bound, force is partitioned between the top ‘loading’ strand (two 12 nt single-stranded regions) and the bottom bridge strand. In the equations shown inset, x designates extension along the loading stand spring. (F) The magnitude of Δx for Rsa4-MIDAS interactions as a function of total applied force. Individual measurements shown as small data points, mean ± SD (n = 11–57 events) shown in bold. Data generated with the 55 and 70 nt bridge constructs shown in red and blue, respectively. Black lines show output of the mechanical circuit model (see also Figure 3—figure supplement 4). (G) The average MIDAS-Rsa4 bond lifetime as a function of force applied across the proteins. Data generated with the 55 nt bridge construct shown in red and data generated with the 70 nt bridge construct shown in blue. Data points shown as mean ± SEM (n = 11–57 events). Black curve shows fit to the catch-slip Bell model.