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 ability of the nitrile to accept a hydrogen bond 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 environment would allow for the direct comparison of environments with and without an extrinsic nitrile probe. Here we incorporate pCNF probes into buried sites near the green fluorescent protein (GFP) fluorophore and measure changes in the electric fields from both the nitriles and the fluorophore in response to a series of amino acid mutations. Changes in electric field measured from both vibrational and electronic reporters are strongly correlated to the change in emission energy of the fluorophore. Additionally, information about side chain chemistry from the series of mutations can be mapped onto the data in a way that links changes in primary structure to the observed fluorescence. Our results also show that pCNF probes can be incorporated near the GFP fluorophore without affecting the electric field changes experienced by the fluorophore. Together these results suggest that GFP provides a rich model system for relating changes in amino acid sequence to observed functional properties. The abundance of water inside the beta barrel of GFP also makes this a useful model system for studying the effect of hydrogen bonding on Stark effect spectra.

Vibrational and electronic Stark effects in green fluorescent protein

251st National Meeting (2016)

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