Supplementary Figure 2: Tight coupling limits fitting of microscopic reaction parameters from ensemble experiments that measure disassembly by light-scattering.
From: Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation
A: Fit (magenta) of a reaction profile (blue) for one experiment using the ‘Simplefit’ script (Supplementary Information). The obtained values of the variables (ka=Hsc70 binding rate; kd=cage disassembly rate; A1=scattering amplitude of the transient Hsc70:cage complex) were used to calculate the overall rate of disassembly (kov) and the time to 50% reaction complete (t1/2) according to the equation: kov=1/(1/([Hsc70]*ka)+1/kd). B: As in A, but kd was fixed at 2x the value in A. Note the large differences in ka and A1 between panels A and B, but the very similar value for kov and t1/2 for both fits. While the fit in A is better, both fits are quite good. This is because a reaction characterized by “fast Hsc70 binding (ka), slow disassembly (kd) and a small scattering amplitude for the transient cage:Hsc70 complex (A1)” will generate a profile very similar to a “slow binding, fast disassembly, large transient scattering amplitude increase” reaction. In practice, we found that experimental variation was greater than the differences in the quality of fits shown above so that microscopic rate parameters like ka, kd or A1 could not be accurately determined from such data. However, the kov values were relatively insensitive to this coupling.
