Photoconversion of the Chromophore of a Fluorescent Protein from Dendronephthya sp.

A. A. Pakhomov, N. Yu. Martynova, N. G. Gurskaya, T. A. Balashova, and V. I. Martynov^*

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia; E-mail: vimart@ibch.ru

^* To whom correspondence should be addressed.

Received December 24, 2003; Revision received April 8, 2004

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A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible light-dependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.

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KEY WORDS: GFP, GFP-like proteins, DsRed, Kaede, fluorescent proteins

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