Many proteins are built from structurally and functionally distinct and domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. Keywords molecular switch; unfolding; natively unfolded; allostery Proteins often display modular architecture that combines protein or small molecule interaction domains with catalytic domains. In such cases, the domains must be coupled, both functionally and structurally, for the protein to attain overall biological activity. For example, ligand binding or phosphorylation can induce structural changes within a regulatory domain that then trigger activity in a catalytic domain. A related type of switching mechanism is illustrated by the recent discovery of proteins that are unstructured in physiological conditions but fold upon binding to their cellular targets. 1,2 Examples include elongin C 3,4 and the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. 5 In these instances, the folding/unfolding of a regulatory domain modulates function of the intact protein via propagation of structural changes. Protein folding makes a particularly effective functional switch because it is reversible and inherently cooperative. Understanding the molecular basis for this type of mechanism is important because it is widely used to regulate protein-protein interactions and in signaling pathways that control cellular behavior. The system consists of a fusion protein in which human ubiquitin (Ub) is inserted into a surface loop of the ribonuclease barnase (Bn) from Bacillus amyloliquefaciens. These proteins were chosen for the following reasons. First, Bn is extremely lethal to both prokaryotic and eukaryotic cells. It is able to be synthesized in B. amyloliquefaciens only because it is co-expressed with its intracellular inhibitor barstar (Bs). 7 This cytotoxic property allows the enzymatic activity...