title: Nitrile Probes of Electric Field Agree with Independently Measured Fields in Green Fluorescent Protein Even in the Presence of Hydrogen Bonding.
abstract: There is growing interest in using the nitrile vibrational oscillation as a site-specific probe of local environment to study dynamics, folding, and electrostatics in biological molecules such as proteins. Nitrile probes have been used extensively as reporters of electric field using vibrational Stark effect spectroscopy. However, the analysis of frequencies in terms of electric fields is potentially complicated by the large ground state dipole moment of the nitrile, which may irrevocably perturb the protein under investigation, and the ability of nitriles to accept hydrogen bonds, which causes frequency shifts that are not described by the Stark effect. The consequence of this is that vibrational spectroscopy of nitriles in biomolecules could be predominately sensitive to their local hydration status, not electrostatic environment, and have the potential to be particularly destabilizing to the protein. Here, we introduce green fluorescent protein (GFP) as a model system for addressing these concerns using biosynthetically incorporated p-cyanophenylalanine (pCNF) residues in the interior of GFP and measuring absorption energies of both the intrinsic GFP fluorophore and pCNF residues in response to a series of amino acid mutations. We show that observed changes in emission energy of GFP due to the mutations strongly correlate with changes in electric field experienced by both the nitrile probes and the intrinsic fluorophore. Additionally, we show that changes in electric field measured from the intrinsic fluorophore due to amino acid mutations are unperturbed by the addition of pCNF residues inserted nearby. Finally, we show that changes in electric field experienced by the vibrational probes trend monotonically with changes in field experienced by the native fluorophore even though the nitrile probe is engaged in moderate hydrogen bonding to nearby water molecules, indicated by the temperature dependence of the nitrile's absorption energy. Together these results demonstrate that even in the presence of hydrogen bonding it is possible to relate nitrile absorption frequencies to electrostatic environment by comparing highly similar environments. GFP's intrinsic linear sensitivity to electric fields makes it a convenient model system for studying electrostatics in proteins that offers lessons for proteins without this visible fluorophore.
authors.affiliation: Department of Chemistry, Center for Nano and Molecular Science and Technology, and Institute for Cell and Molecular Biology, The University of Texas at Austin , 105 E 24th Street STOP A5300, Austin, Texas 78712-1224, United States.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Slocum
authors.affiliation: Department of Chemistry, Center for Nano and Molecular Science and Technology, and Institute for Cell and Molecular Biology, The University of Texas at Austin , 105 E 24th Street STOP A5300, Austin, Texas 78712-1224, United States.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Webb
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D015335:Molecular Probes / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity
chemical_list: D015335:Molecular Probes; D009570:Nitriles; D049452:Green Fluorescent Proteins