250940 Engineering the Beta Roll Domain for Use in Stimulus Responsive Systems

Tuesday, October 30, 2012: 2:18 PM
Westmoreland West (Westin )
Kevin Dooley1, Oren Shur1, Raymond Tu2 and Scott Banta3, (1)Chemical Engineering, Columbia University, New York, NY, (2)Department of Chemical Engineering, The City College of City University of New York, New York, NY, (3)Department of Chemical Engineering, Columbia University, New York, NY

We have extensively characterized a stimulus responsive repeats-in-toxin (RTX) domain and evaluated its potential as a unique scaffold for protein engineering studies.  The RTX domain is intrinsically disordered in the absence of calcium and folds into a beta roll secondary structure consisting of two parallel beta sheet faces in calcium rich environments.   Each beta strand contains two solvent exposed residues that are highly variable in naturally occurring beta roll domains and therefore amenable to mutation.  In one line of work, we have rationally engineered one face to contain leucine residues which facilitate dimerization in the presence of calcium.  This leucine mutant construct can be used as an allosterically regulated cross-linking domain for stimulus responsive protein hydrogel formation.  The conformational change in response to calcium of the leucine mutant has been compared to the wild type through several biophysical techniques including CD spectroscopy, bis-ANS binding, and terbium binding.   The mechanical properties of the mutant beta roll have been characterized through video particle tracking and microrheology.  Secondly, we have used the beta roll domain as a scaffold to evolve novel stimulus responsive binding peptides by using the calcium dependent conformational response to trigger binding and release.  We have created a library of beta roll domains by randomizing eight positions on one beta sheet face.  This library has been expressed monovalently as fusions to M13 phage particles and screened against streptavidin as a proof of concept work.  In the future, we hope to evolve stimulus responsive binding peptides for more useful targets for applications in clinical diagnostics and biosensing.

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