Dwell Time And Maximum Likelihood Analysis Of Single Molecule Disulfide Bond Reduction Events While Under A Stretching Force

Abstract

We study the effects of force on the enzymatic disulfide bond reduction by human thioredoxin (hTRX) in an engineered polyprotein with precise number of disulfide bonds. Single polyprotein molecules are stretched by a cantilever of the Atomic Force Microscope (AFM) in the force-clamp (FC-AFM) mode. Each single disulfide reduction is accurately detected from stepwise increases in the molecule's length vs time (FC traces). Previous FC-AFM studies with E. Coli thioredoxin have proposed two simultaneously occurring disulfide reduction mechanisms producing the overall reduction rate to decrease and then increase with increasing pulling force. In contrast, for the human thioredoxin (hTRX) the overall reduction rate only decreases with a pulling force up to a plateau at forces larger than 300 pN. Here, at each clamping force (100 pN - 400 pN) we collect a large number (> 500 events) of long (>50 s) FC traces. We analyze the data by exponential fits to the ensemble of FC traces and logarithmic histograms of the times elapsed to the actual reduction events (dwell times). Our results demonstrate two force decelerated reduction pathways in 100 pN - 200 pN merging into one apparent pathway in 300 pN - 400 pN. The faster pathway is strongly force dependant and predominates at low forces. The latter one is slower and very weakly force dependant. Next, we apply the maximum likelihood methods (MLM) to fit the FC dwell-time sequences. The MLM confirms the presence of two independent reaction pathways in the whole set of investigated forces. We attribute the faster pathway to a Michaelis-Menten type mechanism with a force-dependant catalytic step. We speculate that the mostly force-independent pathway may represent an electron-tunneling mechanism of reduction.