BIOPHYSICAL STUDIES OF UBIQUITIN: FROM FOLDING TO PROTEIN ENGINEERING

The signaling protein ubiquitin is known for its ubiquity — existing in nearly all cel- lular compartments, holding a prominent role in major cellular signaling pathways and serving as a model system for protein folding. Herein, we honor this stature by exploring several aspects of the ubiquitin system form biophysical, structural, and computational per- spectives. Our efforts begin from the standpoint of protein engineering, where we extend ubiquitin’s function by installing a transition–metal binding motif and elevate it to the sta- tus of a metalloprotein. In doing so, we introduce novel spectroscopic behaviors, reactive propensities, and the capability to form non–canonical polyubiquitin chains — with appli- cations that span from molecular nanotechnology to synthetic biology. We then shift to foundational investigations of ubiquitin’s fold. By characterizing local degrees of freedom, we demonstrate how conformational motions of ubiquitin’s C–terminus can be controlled by the cellular microenvironment. This response, in turn, can regulate molecular recog- nition within the ubiquitination cascade. Finally, we approach global aspects of ubiquitin folding — exploring how a motif containing the C–terminus and the β5 strand might assem- ble into ubiquitin’s β –grasp architecture — with general lessons for ubiquitin–like proteins and other systems with an apparent two–state folding mechanism