Allosteric signaling in proteins relies on a heterogeneous distribution of energetic interactions - the effect of a perturbation at one site (such as ligand binding) is specifically and anisotropically communicated to distant residues at another site (eg. the active site). Recently, a statistical method (Statistical Coupling Analysis, or SCA) has been developed that identifies networks of residues involved in coupling distant functional sites in a large number of protein families. In many cases, mutations of the SCA-identified residues results in disruption or decoupling of protein functions.

A subset of the SCA-identified residues are exposed at the solvent-accessible surface of all protein families studied thus far. These exposed positions offer the opportunity to make post-translational perturbations to the SCA network either by binding of drugs or chemical modification. Our experiment tests the effects of such perturbations at network and non-network surface-exposed residues in Green Fluorescent Protein (GFP). The surface-exposed residues were mutated to cysteine. Further perturbations will then be achieved and spectral properties observed by reacting the thiol side chain with a maleimide reagent.

Results from this experiment show that both network and non-network positions produce bright green fluorescent proteins when mutated to cysteine. This suggests that cysteine mutations are well tolerated at SCA network and non-network positions. The labeling reactions did not produce significant changes in the spectral properties for any of the mutants. In the future, making stronger more directed perturbations may relay more information about the importance of these SCA network positions.