Engineering a Photoactivatable Designed Ankyrin Repeat Protein (2015)

Undergraduates: Alexander Kenan, Ryan Hallett

Faculty Advisor: Brian Kuhlman
Department: Chemistry

---

Designed Ankyrin Repeat Proteins (DARPins) are a class of scaffold proteins that have been engineered to bind to a wide array of protein targets, including caspases and kinases which play important roles in developmental and cellular biology. The goal of this project was to develop a generalizable approach for creating photoactivatable DARPin fusions whose activity could be controlled using light. This inhibition of DARPin activity was to be implemented using the interaction between the Light Oxidative Voltage (LOV) domain and an engineered Z protein that only binds LOV in the dark, disassociating when exposed to light. Short peptide linkers attaching these proteins to the two termini of the protein would then be engineered so that the LOV-Z complex would sterically occlude the binding site of the DARPins in the dark state, but upon light activation would disassociate reactivating the DARPin. Computational modeling and preliminary experimental results demonstrated that native DARPin structures would likely not be caged by a LOV- Z protein interaction due to the orientation of the DARPin termini, and therefore work was done to design and test an anti-parallel helix extension of the C-terminus of the DARPin in order to provide an ideal geometry for the interaction. Testing of these designs by crystallography failed to yield a promising result, but provided insight into future approaches using high throughput techniques for testing designed helices and linker lengths.