GFP Variants with Alternative Strands: Protease Sensor Design and their Thermodynamic Analysis

Abstract

Green fluorescent protein (GFP) variants with one extra strand 10 (s10) can fold with either one or the other s10, and the composition of the two bound forms can be unambiguously distinguished by their resulting colors (Do & Boxer, 2013 JACS). The composition can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have applied thermodynamic analysis on the composition to obtain insight into folding of the variants, and discovered robust empirical rules that accurately predict the product ratios of a given construct. Ratiometric protease sensors were designed from the construct by recombinantly adding a protease cleavage sequence into one of the inserted loops, where the bound s10 is replaced by the other upon protease cleavage and irradiation with light, which is based on a photodissociation phenomenon previously reported (Do & Boxer, 2011 JACS). Since the conversion between the two bound forms involves a large spectral shift, the sensor can display a very high dynamic range. The variants can serve as a platform to provide useful insight into protein folding in general, and further engineering of this class of proteins will enable their application as light-dependent modulators.