Cellobiohydrolase behavior visualized at the single molecule level

Cellobiohydolases, specifically cellobiohydrolase 1 from Trichoderma reesei (TrCel7A), are enzymes that processively degrade cellulose into cellobiose, a glucose dimer. While much work has been done to understand this and other cellulases, a clear understanding of the mechanism underlying cellobiohydrolase motility and degradation has been lacking. Single molecule studies have proven to be instrumental in the discovery of mechanistic behavior of molecular motors, such as kinesin, ClpX, myosin, and RNA polymerase. Here we develop an optical tweezers based motility assay and observe direct stepping of single TrCel7A molecules over distances of one cellobiose unit (~1nm) at a rate of 1 unit every 4 seconds; a result which is largely insensitive to force. We also characterize the behavior of both E. coli expressed catalytic domain and carbohydrate binding module and discover that a delicate balance between binding affinity and motility allows for productive cellulose degradation that only occasionally experiences self-inhibition.