(704d) Engineering the Beta Roll Peptide for Biotechnology Applications

Engineering the Beta
Roll Peptide for Biotechnology Applications

Beyza Bulutoglu¹, Kevin Dooley²
and Scott Banta¹

¹ Chemical
Engineering Department, Columbia University, New York, NY

² Center
for Engineering in Medicine, Harvard Medical School, Boston, MA

In nature, there are many
proteins and peptides that are natively or intrinsically disordered. Often
these peptides gain structure upon interaction with other proteins or
molecules. Using protein-engineering tools, we have explored one of such
peptide: the β-roll. The β-roll is a unique intrinsically disordered,
allosterically–regulated peptide motif. This peptide is isolated from the
repeats-in-toxin (RTX) domain and is intrinsically disordered in the absence of
calcium. In calcium rich environments, the peptide binds Ca⁺⁺ ions
and folds into a β-roll secondary structure composed of two parallel
β-sheet faces. We have extensively characterized this calcium responsive
RTX domain and evaluated its potential as a cross-linker for hydrogel formation
and as an alternative scaffold for biomolecular recognition.

By rationally designing the
two faces of the folded beta helix to contain leucine residues, we have created
an environmentally–responsive cross-linking domain, genetically fused to an α-helical leucine zipper, capable of self-assembly only
in the presence of calcium. We characterized this "double-face" leucine-rich
RTX domain and evaluated its mechanical and biophysical properties using
various techniques such as circular dichroism and michrorheology. To further
investigate its cross-linking capability, we constructed concatemers of this
β-roll with maltose binding protein (MBP) and demonstrated that the
engineered β-roll peptide can mediate calcium-dependent proteinaceous
hydrogel formation without the need for other cross-linking moieties.

In addition, we proposed
that the β-roll peptide would be a suitable binding scaffold combining
molecular recognition with a simple elution mechanism. This will be of great
interest in the bio-separation area as well as other areas of biotechnology, where
binding events can be controlled by benign environmental perturbations.
Engineering the β-roll results in an allosterically–regulated
scaffold, where the folding function can be decoupled from the binding
function. Thus, this peptide can operate as a protein switch for biomolecular
recognition, which can be mediated by simply changing the calcium
concentration, allowing control over the binding behavior of the molecules.

For these purposes, we
constructed a library containing mutated residues in eight positions, which
form the beta sheet face upon folding. This way, one face of the folded
β-roll is fully randomized. Using the ribosome display technique, we
selected new β-roll peptides showing affinities for various targets
including lysozyme and the Fc region of an IgG antibody. Isothermal titration
calorimetry (ITC) was utilized to quantitatively determine the thermodynamic
parameters of interactions in solution between the mutants and their targets.
The latest results on the molecular recognition capabilities of the candidates
obtained via this method will be presented.