Intrinsically disordered peptides do not adopt a well-defined native structure. Upon interaction with small molecules or other peptides, they undergo induced folding. Using protein engineering tools, we have explored one of such peptide: the β-roll. In the absence of calcium, it is intrinsically disordered and it folds upon calcium binding, gaining a secondary structure composed of a beta helix with two parallel beta sheets. We have extensively characterized this peptide and evaluated its potential as a cross-linking domain capable of hydrogel formation in the presence of calcium only.<br/><br/>Recently, we started exploring the potential of this peptide as an alternative scaffold for biomolecular recognition. We created a library of mutant β-rolls, where we randomized one face by mutating the residues, which form the beta sheet face upon calcium binding. Ribosome display technique was employed to capture promising mutants, which demonstrate an affinity for various targets including lysozyme and Fc domain of an antibody. Promising candidates were evaluated via isothermal titration calorimetry, where we quantitatively determined the thermodynamic properties of interactions of the mutant β-rolls. 10 beta roll peptides were downselected from a library and 3 were found to have a better affinity towards lysozyme compared to WT β-roll, with low-micromolar dissociation constants. We also employed different strategies to improve the binding affinity such as cloning concatemers of mutant β-rolls, where we achieved high-nanomolar dissociation constants.<br/><br/>Throughout this study, the β-roll peptide is explored as an allosterically-regulated scaffold, where the folding and binding function can be decoupled. By employing this peptide as a protein switch for biomolecular recognition, the binding behavior of the molecules can be controlled by simply adjusting the calcium concentration. Evolving such an intrinsically disordered peptide to demonstrate affinities for various molecules can provide useful applications in the bioseparations area as well as other areas of biotechnology.

Evolving an intrinsically disordered peptide, the β-roll, for biomolecular recognition

251st National Meeting (2016)

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