By Tien Do, Chemistry; Yasan Fonseka, University of Cincinnati
Advisor: George Stan
Abstract: Bacterial caseinolytic proteases(Clp) nanomachines support protein quality control through the degradation of misfolded or excess proteins which helps to maintain protein homeostasis. These hexameric nanomachines utilize cyclical allosteric motions to apply mechanical force onto the Substrate Protein(SP) that enables SP unfolding and translocation through the central pore of the machine. Haloalkane Dehydrogenase (Halo Tag) is a good candidate to study the unfolding and translocation mechanisms due to its biological and experimental importance. Topologically, HaloTag is an _-helix rich bulk protein that consists of a central buried-sheet registry. To study the mechanisms of the unfolding of HaloTag, we performed Langevin molecular dynamics coupled with targeted molecular dynamics(TMD). Here we used the Effective Energy Function (EEF1) implicit solvation model to represent the proteins. Our simulation setup consists of restrained or unrestrained geometries that mimic the experimental (Laser Optical Tweezers/ Atomic Force Microscopy) and in vivo degradation mechanisms respectively. We observe that, in the unrestrained geometry, ClpY nanomachine assists the HaloTag to orient in a direction that favors the remodeling of the SP but this process is stalled upon formation of a large number of non-native contacts from the _ helices, specifically at _4 and _8. In the restrained geometry, SP rotation about the axial direction enables an efficient mechanism of removal of exposed _ helices that overcomes the large number of non-native contacts.