Project Summary/Abstract Intracellular AAA+ proteases remove toxic, damaged, and unnecessary proteins and participate in cellular processes that promote health and prevent disease. Because protein synthesis is energetically expensive and proteolysis is irreversible, protein degradation must be carefully regulated to conserve cellular resources. Building on our extensive experience in structure-function studies of AAA+ proteases, we will use a combination of structural biology, biochemistry/biophysics, protein engineering, and molecular genetics to determine how the proteolytic activity of three different AAA+ proteases is mediated and regulated. For example, for the AAA+ HslUV protease, we will determine how temperature and substrate binding control autoinhibition by a novel I-domain structure that blocks the axial pore. For the double-ring ClpAP protease, we will probe the roles of the AAA+ D1 and D2 rings in protein substrate unfolding and translocation in single-molecule optical trapping experiments. For FtsH, an essential membrane-bound bacterial AAA+ protease, we will determine the mechanisms that limit degradation of cytosolic proteins, mediate turnover of an important biosynthetic enzyme, and allow the HflK/HflC membrane partners to control FtsH activity. The unifying theme of this proposal is understanding how the common and unique structural features of different AAA+ proteases contribute to their specialized biological roles. !