The overall goal of this innovative and high risk NSF EAGER award, funded by the Biotechnology, Biochemical and Biomass Engineering Program and by the Biosensing Program, is to use directed evolution to modify novel calcium-regulated beta roll peptides so that they can bind to protein targets with built-in allosteric recognition. The beta roll peptide has a cork-screw structure that reversibly forms in the presence of calcium. This domain has not evolved to bind to other proteins, but structurally it resembles the leucine rich repeat and ankyrin repeat proteins, which have both evolved, and been engineered, for biomolecular recognition. The researchers have begun to use directed evolution to create new beta roll peptides that bind to a model protein target, and the built-in allosteric regulation of the scaffold will enable the binding interface to be formed depending on the calcium concentration. This will be the first example of the directed evolution of an intrinsically disordered peptide for biomolecular recognition. These peptides can then be used in sensing or other molecular engineering applications.
Most proteins have defined three-dimensional structures and protein engineers have developed tools and techniques to engineer many aspects of the molecules for different applications including their ability to bind other target molecules. This biomolecular recognition is important in applications including therapeutics, separations, biosensors, etc. Nature uses folded proteins with well-defined structures for this purpose, however it is becoming increasingly clear that some proteins do not have native, well-defined structures. These intrinsically disordered proteins and peptides have dynamic structures but can adopt a conformation when they bind to a target molecule. The central goal of this exploratory grant was to develop a method to engineer an intrinsically disorder peptide to bind to a target protein. The RTX domain is found in some secreted bacterial proteins and it is intrinsically disordered in solution. However, in the presence of calcium it reversibly folds into a flattened cork-screw conformation called a beta roll. This domain is found in many larger native proteins and it is not known to be involved in biomolecular recognition. We set out to see if we could take a native beta roll peptide and engineer it to bind to an arbitrary target protein. The idea would be that the peptide would bind the target in the presence of calcium when it adopted the beta roll structure. In the absence of calcium the engineered peptide would be intrinsically disordered and thus unable to bind to the target. We have now successfully developed a platform using in vitro protein expression of a partially randomized beta roll library and we have selected this library over immobilized lysozyme proteins. After several rounds of panning, we were able to find a beta roll sequence that has affinity for lysozyme solely in the presence of calcium. Thus, this is the first example of an intrinsically disordered peptide that has been engineered for biomolecular recognition. This advance sets the stage for the future development of this project: to fully characterize this biomolecular interaction, to increase the affinity of the binding, and to evolve beta roll peptides with high affinity to other targets of biotechnological importance.
