Chromophore-Protein Interactions in the Anthozoan Green Fluorescent Protein asFP499

Abstract

Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The x-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 A, shows the typical beta-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln(63)-Tyr(64)-Gly(65), is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The x-ray structure provides interesting clues as to how the spectroscopic properties are fine tuned by the chromophore environment.

Blue light regulation of host pigment in reef-building corals

“…Fluorescent proteins (FPs) emit light in the color range from cyan to red (Wiedenmann 1997, Matz et al 1999, Wiedenmann et al 2000, 2004, Dove et al 2001, Shagin et al 2004, Wiedenmann & Nienhaus 2006, Oswald et al 2007), whereas chromoproteins (CPs) display bright purple to blue colors but are non-fluorescent (Wiedenmann et al 1999, Lukyanov et al 2000, Dove et al 2001, Shagin et al 2004. In contrast to GFP, its anthozoan homologues usually form homotetramers (Nienhaus et al 2003(Nienhaus et al , 2006a. In recent years, proteins from the GFP family have become powerful tools in biomedical research.…”

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

“…1. Cyan fluorescent proteins such as asFP499 are frequently found in reef building corals and sea anemones (19,28,29). They feature the chemically unaltered p-HBI chromophore of GFP.…”

“…They feature the chemically unaltered p-HBI chromophore of GFP. Polar interactions of the chromophore and its surrounding residues affect the charge distributions in the ground and electronically excited states and result in blue shifted emission (29)(30)(31)(32). The yellow emission of zFP538 from Zoanthus sp.…”

Photoactivation in green to red converting EosFP and other fluorescent proteins from the GFP family

“…By contrast, most anthozoan GFPs show maximum absorption around 480 -500 nm at neutral pH, indicating that their chromophores exist preferentially in the anionic B state. Exceptions are asFP499 from A. sulcata 23,50 and cgigGFP from Condylactis gigantea 51 , which both show a dominant excitation band at ~400 nm. The absorption of the anionic B form of the chromophore of EosFP is characterized by an extinction coefficient ε(506 nm) = 72,000 M -1 cm -1 ; the fluorescence quantum yield was determined as 0.70 ± 0.02.…”