Supercharging as a General Strategy for Making Proteins into Conformational Switches and their Use in Biosensing

Abstract

Greatly increasing the magnitude of a protein's net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4 using simple molecular modeling to create a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction in biology. To demonstrate this process using more complex proteins, we then modified green fluorescent protein and the cytochrome B562, using a combination of simple modeling and electrostatic calculations to create proteins that unfold reversibly with decreasing ionic strength. These simple model systems allow us to derive and then experimentally validate a mass-action model for the coupled folding and binding behavior of ligand-gated conformational switches, establishing a set of engineering principles which can be used to convert natural and designed soluble proteins into molecular switches useful in biodesign and synthetic biology. We have applied these principles to a biosensing project in which supercharged IDPs are attached to nanostructured gold surfaces and conformational changes are sensed using surface plasmon resonance. An increase of the refractive index at the gold surface is caused by the ligand induced conformational change and can be detected via transmission surface plasmon resonance (SPR) spectroscopy. We calculate that the shift in the resonance wavelength of the surface plasmons is almost two orders of magnitude more than simple ligand binding to an already folded protein. Continuing SPR studies provide practical insight into the use of our model as conformational switches for biosensing devices.