Fig. 4.

Bond lifetimes of beads functionalized with different streptavidin variants. (A) Survival fraction as a function of time for commercially available beads coated with tetravalent streptavidin (Dynabeads M-270 Streptavidin; Invitrogen), tethered by ddFLN4 and subjected to a constant force of 65 pN. The survival fraction decays in a complex, multiexponential fashion, suggesting the existence of several populations of biotin–streptavidin interactions with different lifetimes. Red and black lines are linear fits to the logarithm of the first and last 20% of data points, respectively, to estimate off-rates of the fastest-decaying (A, Inset) and slowest-decaying populations. (B) Survival fraction as a function of time for ddFLN4-tethered beads functionalized with a monovalent streptavidin variant, measured at 65 pN. The blue line is a linear fit to the natural logarithm of all data points. Note the markedly different scale of the time axis. (C) Estimated lifetime of the biotin–streptavidin interaction at different constant forces for the fastest- and slowest-decaying population of commercial beads with tetravalent streptavidin (red and black circles, respectively) and for beads with monovalent streptavidin (blue triangle). Error bars correspond to 1 SD; lines are fits of a single-barrier kinetic model. The number of measured, specifically tethered beads per condition was between 44 and 118, of which 35–86 ruptured during the duration of the measurement. The measurement duration was 15 h for beads with monovalent streptavidin and for the commercial beads 1 h at 65 pN, 3 h at 55 and 60 pN, and 15 h at 45 pN.