Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein

Abstract

dene)-5-imidazolinone, by nucleophilic attack of Gly 67 -N␣ on the carbonyl of Ser 65 , dehydration, and oxidation of The Institute of Physical and Chemical Science (RIKEN) 2-1 Hirosawa, Wako-city the ␣-␤ bond in Tyr 66 (Heim et al., 1994; Tsien, 1998; Reid and Flynn, 1997). One example of GFP-like protein Saitama 351-0198 Japan is a red-emitting fluorescent protein, DsRed (Matz et al., 1999). DsRed fluoresces first green and then red,

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

“…4 A and B), whereas the band at Ϸ25 kDa representing the full-length peptide decreased. Apparently, green-to-red conversion involves dissociation of the peptide backbone, as was also observed for Kaede (14,15). The 20-kDa band contains the fluorophore, as seen from its fluorescence after transfer to a nitrocellulose membrane (Fig.…”

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

“…This spectral change arises from an extension of the chromophore, which is accompanied by a break in the backbone between Phe-61 and His-62 [22]. This irreversible modification allows the delocalized π-electron system to also include the His-62 imidazole sidechain [24], thereby creating a red-shifted chromophore. Pronounced vibronic sidebands, well separated from the main peak, are observed in the redshifted species at 553 nm in the excitation spectrum and at 629 nm in the emission spectrum.…”

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

“…Cyan and green fluorescent proteins contain a chromophore chemically identical to that of GFP 5 while yellow, orange, and red emission results from additional modifications to the polypeptide backbone adjacent to a GFP-like chromophore. [6][7][8][9][10][11][12][13][14] In DsRed, the peptide bond immediately preceding the chromophore is oxidized to an acylimine; consequently the conjugation of the chromophore extends over the polypeptide backbone, increasing both the excitation and emission maxima relative to GFP. 6,7 zRFP574 14 and eqFP611 contain a DsRed-like chromophore although in the latter case, the chromophore adopts a trans conformation instead of the more typical cis conformation.…”

“…9 In Kaede and EosFP, light-driven backbone cleavage results in extension of the conjugation of a GFP-like chromophore to the imidazole ring of the histidine in the chromophore-forming tripeptide, leading to a green to red shift in emission. 8,10,13 Further modifications to the acylimine of a DsRed-like chromophore also occur in several fluorescent proteins. In the ''kindling fluorescent protein'' (KFP), hydrolysis of the acylimine linkage was proposed, resulting in backbone cleavage and the introduction of a new carbonyl group conjugated with the chromophore.…”