This proposal seeks to support an ongoing Iongterm project concerning the mechanism of action of the essential megadalton-sized double ring molecular machines known as chaperonins, which mediate ATPdependent protein folding to the native state in a variety of cellular compartments. Here, we propose studies of the bacterial chaperonin, GroEL, and its cooperating co-chaperonin, GroES, both in vivo and in vitro. One study in vivo will aim to resolve the physiological action of GroEL, not well-understood due to lack of a tight conditional mutant, by identifying a chemical compound that binds to the GroEL ATP binding pocket and produces immediate-onset loss of function in vivo. We will examine whether the protein translation machinery is halted by such arrest of the GroEL folding machine, and will inspect for misfolding/aggregation both of nascent polypeptides, if they are produced, and of pre-existent proteins. This will allow us to observe the immediate physiologic consequences of disruption of the chaperonin system and should identify its authentic and essential substrates. In a second area, mechanistic studies will be carried out in vitro, following on our earlier studies of the machine itself, directing analysis to the polypeptide substrate. We will analyze the global topology of substrate proteins while bound to GroEL, using oxidative crosslinking between native and engineered cysteines in the substrates themselves, addressing whether the topology is unique and potentially native-like, random, or a limited ensemble of states. We will also address whether bound polypeptides occupy a characteristic topology relative to GroEL, with a particular portion bound inside the central cavity of a GroEL ring while the remainder is localized outside in the bulk solution, using external labeling of the exposed portion of substrate protein and single particle imaging with cryoEM. We will also address actions of nucleotide/GroES binding on GroEL-substrate binary complexes. One study will use cryoEM to examine complexes formed by addition of ADP/GroES to GroEL-rhodanese, in which apical domain movement is severely retarded, potentially providing a structure of a GroEL-GroES collision complex. In a second study, the question of whether ATP/GroES binding to GroEL-substrate complexes produces an active, forced, unfolding of substrate protein prior to its release into the central cavity will be studied using tritium and hydrogen/deuterium exchange experiments. [unreadable] [unreadable]