Vibrational and Electronic Stark Effects in Green Fluorescent Protein

Abstract

The use of unnatural amino acids to probe non-covalent interactions, dynamics, and folding in proteins is growing rapidly. Specifically, para-cyanophenylalanine (pCNF) is an attractive probe of electrostatics because of its sensitivity to electric fields and its minimal structural perturbation when replacing phenylalanine or tyrosine. However, due to the potential for hydrogen bonding to the nitrile moiety there is potential for the introduction of nonnative interactions, which can cloud our understanding of native proteins. Usual attempts to quantify the extent of nitrile perturbation include functional assays or structural measurements. While structure and function are undoubtedly linked to electrostatic environment, they are not direct reporters of it. An intrinsic reporter of electrostatic environment would allow for the direct comparison of environments with and without an extrinsic nitrile probe. Boxer and coworkers have shown that the intrinsic fluorophore of green fluorescent protein (GFP) is sensitive to electric fields in a manner consistent with a linear Stark effect. Here we incorporate multiple pCNF probes into buried sites near the GFP fluorophore and measure changes in the electric field environment from both the nitriles and the fluorophore in response to a series of amino acid mutations. Our results show that pCNF probes can be incorporated near the GFP fluorophore without affecting the electric field changes experienced by the fluorophore. Additionally, changes in electric field measured from the nitrile are in agreement with those measured from the fluorophore. These results suggest that, for this model system, nitrile probes do not significantly perturb the native electrostatic environment. The agreement of changes in electric field from vibrational and electronic Stark effect probes inside the beta barrel of GFP suggests nitrile frequencies can be interpreted in terms of the Stark effect even when they participate in hydrogen bonding.