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Studying Protein Electric Fields in GFP

Two types of field probe,
Neighbors in a green barrel;
Don't know they're different.

-Josh Slocum

Electrostatic fields generated by the three-dimensional distribution of partial charges throughout a structured protein provide a fundamental link between amino acid sequence and function, including folding, reactivity, kinetics, and macromolecular interactions.  Understanding this link would enable the design of enzymes with improved functions, combat disease states, and provide benchmarks for computational models that accurately predict electostatic fields. Our laboratory uses vibrational Stark effect (VSE) spectroscopy of nitrile probes covalently inserted into proteins to directly measure electric fields in a wide range of biological contexts. 

The nitrile stretching oscillation has been widely used as a probe of local environment to study dynamics, folding, and electrostatics in proteins.  However, the interpretation of nitrile frequencies in terms of electric fields is complicated by the fact that hydrogen bonding to the functional group also causes frequency shifts that are not described by the Stark effect.  Additionally, the large ground state dipole moment of the nitrile itself could possibly change the local electrostatic environment that it is being used to measure. 

Strategy: To address these basic concerns, the Webb group has biosynthetically incorporated para-cyanophenylalanine (pCNF) residues into green fluorescent protein (GFP) near its intrinsic chromophore, whose sensitivity to electric fields has been well characterized. Measurements of absorption energy changes of the nitrile probe and the intrinsic fluorophore in response to a series of nearby amino acid mutations show that the vibrational and electrostatic probes respond similarly to the same mutations, and that the intrinsic sensitivity of GFP emision energy to these mutations is unperturbed by the presence of the nitrile probes themselves.      

We are using this model system to asses all aspects of nitrile vibrational shifts in proteins. We are also using our experimental data to test and refine computational strategies for predicting electric fields in proteins of known structure, in our own laboratory and with collaborators developing novel and innovative methods to understand all aspects of the photophysics of nitrile probes in complex environments.

People:  Josh Slocum and Desiree Fernandez

Blasiak, B.; Ritchie, A. W.; Webb, L. J.; Cho, M.  "Vibrational Solvatochromism of Nitrile Infrared Probes: Beyond Vibrational Stark Dipole Approach."  Phys. Chem. Chem. Phys. 2016, DOI: 10.1039/c6cp01578f.

Slocum, J. D.; Webb, L. J.  "Nitrile Probes of Electric Field Agree with Independently Measured Fields in GFP Even in the Presence of Hydrogen Bonding."  J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b02156.