Large negative standard heat capacity changes (ACp << 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977)

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Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation—which can be carried out by a molecule as small as a thousand atoms or so—belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, bio-technology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and “smart” biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding–unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.

The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.

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...