470417 Engineering New Allosterically - Regulated Protein Interactions for Applications in Biotechnology

Thursday, November 17, 2016: 1:06 PM
Continental 7 (Hilton San Francisco Union Square)
Beyza Bulutoglu and Scott Banta, Department of Chemical Engineering, Columbia University, New York, NY

In nature, there are many proteins and peptides that exhibit allosteric regulation. These natively disordered proteins gain structure when they interact with other proteins or small molecules, such as ions. Using protein-engineering tools, we have investigated one of such peptide: the β-roll. This peptide is isolated from the Block V repeats-in-toxin (RTX) domain of Adenylate Cyclase from Bordetella Pertussis. It is disordered in the absence of calcium and it folds into a β-roll secondary structure composed of two parallel β-sheet faces upon binding to Ca++ ions. This way, the peptide can transition between its unfolded state and the β-roll structure in a reversible way. We have utilized the allosteric regulation of this domain as a tool to engineer new protein-protein interactions. In its folded state, the peptide has two faces, serving as binding surfaces available for interaction with other proteins. Each beta strand in the β-roll face has two residues with solvent exposed side chains. Our work involves alteration of the residues, which form these faces upon calcium binding, via combinatorial protein design techniques.

We evaluated the potential of this peptide as a cross-linker for hydrogel formation. We showed that calcium dependent hydrogel formation can be achieved by designing the two faces of the folded beta helix to contain leucine residues. When there is no calcium, β-roll domains remain unstructured, delocalizing the leucine rich patches. After calcium binding, the β-roll folds and the leucine rich faces are exposed creating a hydrophobic driving force for self-assembly1. We created various constructs where we used the leucine rich β-roll mutant as a hydrogel building block. In addition, we showed that this peptide can serve as a new scaffold for biomolecular recognition. We randomized the amino acid residues on one of the beta sheet forming faces and selected mutants, which demonstrated an affinity for various targets, including lysozyme. Isothermal titration calorimetry (ITC) was utilized to quantitatively determine the thermodynamic parameters of interactions in solution between the mutants and their target. In the case of the lysozyme library, we achieved sub-micromolar / mid-nanomolar (0.52μM - 63nM) dissociation constants. We also performed affinity chromatography experiments and showed that the best mutant is capable of capturing its target, in the presence of calcium2. The reversibility of the calcium binding allows for the engineered molecular recognition to be controllable. The captured target is easily eluted upon removal of the calcium ions. This will be of great interest in the bio-separation area as well as other areas of biotechnology, where binding events can be easily and reversibly controlled.

References:

  1. K. Dooley, B. Bulutoglu and S. Banta, Doubling the cross-linking interface of a rationally-designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation. Biomacromolecules, 2014, 15(10): 3617-3624.
  2. B. Bulutoglu, K. Dooley, M. Blenner and S. Banta, Catch & Release: Fishing for target proteins with an evolved β-roll peptide exhibiting allosteric regulation, submitted.

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