Date of Graduation


Document Type


Degree Type



School of Medicine



Committee Chair

David M. Smith

Committee Co-Chair

Michael D. Schaller

Committee Member

Maxim Sokolov

Committee Member

Michael R. Gunther

Committee Member

David P. Siderovski


Across all domains of life, the proteasome is responsible for the majority of targeted protein degradation in the cell. Often, the proteasome is thought of as the molecular “garbageman” of the cell. While it is true that the proteasome degrades and eliminates misfolded proteins, the proteasome is also capable of degrading fully folded, functional proteins whose presence is no longer required (e.g. during embryonic development, cell cycle changes, etc.). Despite its crucial role in virtually every cellular process, our understanding of how the proteasome operates from a mechanistic perspective is still highly limited. In order to prevent unregulated degradation the protease sites of the proteasome are sequestered inside its hollow interior. While loosely folded proteins can enter the degradation chamber without the requirement of energy, proteins with secondary structure can only be degraded when they are properly recognized (e.g. by ubiquitin tags), unfolded, and injected into the protease chamber for degradation. Protein recognition, unfolding, and injection into the protease chamber all depend on ATP. However, very little is known about how such chemical energy is converted to mechanical work. In this dissertation we sought to understand the logistics of nucleotide binding and hydrolysis, and also to determine the conformational changes that can regulate protein entry and degradation by the proteasome. To this end, we focused on one of the most common regulators from eukaryotes-- the heterohexameric 19S ATPases, as well as its homohexameric archaeal homolog-- “PAN” (proteasome activating nucleotidase). Based on our extensive analysis, our data support a neighbor-binding sequential hydrolysis mechanism for the proteasomal ATPases. Furthermore, we show that these ATPases are highly processive, even when they reach more tightly folded domains of a protein, which is unlike what had been proposed previously based on studies of other ATP-dependent proteases (e.g. ClpX, which often “slips” and “stalls” at these more tightly folded domains). This tight binding of the proteasomal ATPases appears to be due to its crucial trans-arginine fingers that “sense” bound nucleotides in its neighboring subunit (which ClpX lacks), and we propose that this processivity arose due to the diverse client proteins the proteasome must encounter (e.g. it must engage and unfold many types of proteins, even ones that are fully folded and functional). Lastly, we have developed a disulfide engineering approach to show that PAN’s N-terminal domains adopt distinct conformations that set the rate of ATP hydrolysis. This novel approach has allowed us to isolate specific subunits from a homohexamer that are identical in their amino acid sequence, but that adopt different conformations when they form a hexameric ring. This disulfide engineering approach we’ve developed is a powerful method to analyze structural asymmetries in homomeric protein complexes with minimal structural perturbations, which has not been accomplished before and opens the door to an entire new approach to studying the function of the molecular motors. We started this work with the goal of understanding the logistics of the mechano-chemical cycle of the proteasomal ATPases. Indeed, we have developed novel methods to better understand the inner workings of this complex multimeric machine, and the groundwork we lay here has contributed greatly to our knowledge of the proteasomal ATPases, and will also push forward our understanding of other AAA+ ATPases and molecular motors in general. Ultimately, a better understanding of these complex machines will aid in the development of new therapies to combat diseases where these machines are dysregulated.