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Probing the chromophore of the green fluorescent protein with 4‐cyano‐L‐phenylalanine
Author(s) -
Piacentini Juliana,
Olenginski Gregory M.,
Brewer Scott H.,
PhillipsPiro Christine M.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.602.2
Subject(s) - chromophore , green fluorescent protein , chemistry , fluorescence , fluorescent protein , phenylalanine , amino acid , solvation , isotopomers , photochemistry , molecule , biochemistry , organic chemistry , physics , quantum mechanics , gene
The unnatural amino acid (UAA) 4‐cyano‐L‐phenylalanine (pCNF) has been shown previously to be an effective vibrational reporter of local protein environments. Here, pCNF was genetically incorporated into the 66‐site of superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl‐tRNA synthetase to probe the local environment around the GFP chromophore. The 66‐site is in the internal chromophore of the protein, which is responsible for the photo‐physical properties of sfGFP. Vibrational spectroscopy coupled with isotopomers of this UAA was utilized to probe the local solvation state of this site. This spectroscopic data was coupled with the X‐ray protein crystal structure of this construct. The spectroscopic and crystallographic data will be presented.