The structure of a protein is closely related to the function of that molecule, hence our interest in the relation between the non-covalent interactions which give rise to the overall structure of a protein. We measure non-covalent interactions using the model system Green Fluorescent Protein (GFP). Experimentally, we are able to make perturbations to a wild type protein at specific residues causing energetic changes experienced by the protein chromophore, and by nitriles integrated via nonsense-suppression. The changes in frequency of strategically placed nitrile probes, in close proximity to the protein GFP chromophore, are measured by IR absorbance spectra and a UV Vis absorbance spectra shifts; thus, indicating a change in protein electrostatics. Nitriles are particularly useful as probes in our system due to their sharp absorption peak in the range of infra-red (IR) incidence radiation which is outside the range of the background protein absorbing in the UV-visible light region.<br/>Here we will show recent work with a cystine mutant that has yielded data which suggests that the nitrile and chromophore are susceptible to electrostatic changes caused by deprotonation of the cystine residue. Not only does the chromophore demonstrate evidence of sensitivity to local electric field changes, but there are two distinct changes occurring near the chromophore. Additionally, due to its close proximity, the nitrile also appears to be sensitive to the protonation and deprotonation states of the cystine residue.

Experimental measurements of protein electrostatics in GFP using vibrational stark effect spectroscopy

253rd National Meeting (2017)

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