Evolving the Beta Roll Domain for Regulated Molecular Recognition
Molecular recognition is ubiquitous in nature. Frequently antibodies are used in technology applications where biomolecular recognition is to be employed, but antibodies have several limitations in these applications, including difficulty in easily removing bound targets. This becomes especially important in the development of biosensors using electrochemical-based signal transduction schemes.
Instead of trying to engineer allosteric control into a molecular recognition molecule, we have started with intrinsically disordered scaffold, the beta roll domain, and we are working to evolve this allosterically regulated scaffold for biomolecular recognition. The naturally existing beta roll subdomain motif consists of tandem repeats of the sequence GGXGXDXUX, where U is an aliphatic amino acid and X is any amino acid. In the presence of calcium, the disordered peptide reversibly transitions to a beta roll spiral structure of two parallel beta sheet faces, where each beta strand has two solvent exposed variable residues.
We have characterized a native beta roll subdomain with various end-capping groups in order to identify a minimal calcium-responsive beta roll unit. We have immobilized the beta roll on surfaces, and we have developed a FRET-based system to monitor structural perturbations. We have truncated and concatenated the beta roll and we have discovered beta roll sequences that can reversibly precipitate which is useful for protein purification. We believe that the beta roll faces are suitable binding surfaces and that calcium-induced structure formation can be used as a mechanism to control the formation of the engineered biomolecular recognition interface. To test this, we have engineered beta rolls to dimerize in a calcium-dependent manner and used these to create cross-links for protein hydrogels. We have randomized one face of the beta roll unit and we are using directed evolution to identify beta roll peptides with biomolecular recognition capabilities.
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