Szoszkiewicz, Robert2013-04-182013-04-182013-04-01http://hdl.handle.net/2097/15526Disulfide bond reduction pathways used by human thioredoxin (hTrx) are studied at the single molecule level using a recombinant protein (I27[subscript SS])[subscript 8]. (I27[subscript SS])[subscript 8] contains eight tandem repeats of identical immunoglobulin-like modules with one disulfide bond in each module. Single (I27[subscript SS])[subscript 8] molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Disulfide reduction events are accurately detected from stepwise increases in the end-to-end length of (I27[subscript SS])[subscript 8]. Earlier FC-AFM studies observed one disulfide reduction pathway used by hTrx and suggested an additional electron tunnelling mechanism. Here, a very large set of unbiased FC-AFM data is collected in a range of clamping forces. By analyzing the data using exponential fits and dwell times histograms two disulfide reduction pathways used by hTrx are resolved. Based on previous studies one of these pathways is attributed to force-dependent Michaelis-Menten catalysis. The latter reduction pathway is weakly force-inhibited and occurs sporadically. Bimolecular nucleophilic substitutions (S[subscript N]2) and electron tunnelling (ET) mechanisms are discussed to explain the second pathway. Direct S[subscript N]2 and ET mechanisms cannot be discounted, but a hypothetical E2-S[subscript N]2 mechanism involving a hydride reducing a disulfide bond provides an interesting alternative, which needs to be verified in future experiments.en-USThis Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).Atomic force microscopySingle molecule biophysicsProtein chemistryDisulfide bond reductionThioredoxinEnzyme kineticsEnsemble kinetic analysisDwell time histogramsArticle (author version)