(307d) Engineered Multi-Input Protein Switches As Versatile Biosensors with Target-Specific Controls

Engineered multi-input protein switches as versatile biosensors with
target-specific controls

Jay H. Choi and Marc Ostermeier

Department of Chemical and Biomolecular Engineering, Johns Hopkins
University, Baltimore, Maryland

Protein
switches are engineered proteins that are composed of an input domain that
recognizes and responds to an input signal and an output domain whose function
is regulated by the state of the input domain.  Engineered
protein switches have a number of exemplary properties for sensing applications
including a large dynamic range, high specificity for the activating ligand,
and a modular architecture that will facilitate fine-tuning of the desired
properties.  Thus, protein
switches can provide a unique platform for the next generation biosensor systems.
 The modular regulation mechanism of protein switches allows the possibility
of developing versatile protein switches, in which different input domains can
be coupled to the same output domain.  However, switches engineered to
date are limited to have natural ligands or similar biomolecules as input
signals.  For the development of versatile biosensors, it would be
desirable to have input signals that can be easily controlled or to have multiple
input signals that can be manipulated independently.  Especially in
cellular environment, multi-regulated switches is highly desirable for the
target specific control.  We have developed protein switches that can be simultaneously
regulated by multiple input signals including small molecules, temperature, pH,
and redox potential.  These protein switches are designed and constructed
based on a platform of a previously developed small molecule activated protein switch. 
We have taken advantage of its modular regulation mechanism that controls the
activation of enzyme in the output domain by manipulating active conformational
states.  These protein switches function via the dual regulation mechanisms
such that they are pre-activated by changes in temperature, redox potential, or
pH, and then subsequently activated by small molecules.  Thus, the
engineered multi-regulated protein switches require dual input signals for the
complete activation of enzyme.  This design principle has also been
applied to develop reversely regulated protein switches.