title: Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy.
abstract: We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).
authors.affiliation: Department of Chemistry, Graduate Program in Biophysics, and Graduate Program in Molecular and Cellular Biochemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400, USA.
authors.forename: A F
authors.initials: AF
authors.lastname: Bell
authors.forename: X
authors.initials: X
authors.lastname: He
authors.forename: R M
authors.initials: RM
authors.lastname: Wachter
authors.forename: P J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D000085:Acetates / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D019943:Amino Acid Substitution / Q000235:genetics; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D003373:Coumaric Acids; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009584:Nitrogen / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011489:Protein Denaturation; D011522:Protons; D012997:Solvents; D013059:Spectrum Analysis, Raman
chemical_list: C403206:4-hydroxycinnamoyl-CoA; D000085:Acetates; D000214:Acyl Coenzyme A; D000838:Anions; D002021:Buffers; D002412:Cations; D003373:Coumaric Acids; D005456:Fluorescent Dyes; D007093:Imidazoles; D008164:Luminescent Proteins; D011522:Protons; D012997:Solvents; C409202:ethyl 4-(4-hydoxyphenyl)methylidene-2-methyl-5-oxoimidazoleacetate; D049452:Green Fluorescent Proteins; C022903:cinnamoyl-coenzyme A; D044943:Fatty Acid Desaturases; D042964:Acyl-CoA Dehydrogenase; D009584:Nitrogen; D010100:Oxygen