The in vivo role of GFP

Several bioluminescent coelenterates such as the jellyfish Aequorea victoria or the sea pansy Renilla reniformis emit green light from a green fluorescent protein (GFP). David Galbraith has mounted a beautiful picture of A. victoria.

The GFP's serve as energy-transfer acceptors, receiving energy from a Ca2+-activated photoprotein or a luciferase-oxyluciferin complex in A. victoria and R. reniformis, respectively.

Upon mechanical stimulation A. victoria emits a green light. However, when the calcium-triggered photoprotein aequorin was puriefied from A. victoria photocytes, it was found to generate blue, rather than green light. GFP acts as a secondary fluorescent protein, receiving energy from activated aequorin. It has been suggested that radiationless energy transfer is involved, but this subject seems still under discussion [3].

The following image summarizes A. victoria bioluminescence [10]:

Aequorin (shown on the very left) is a complex of the 21.4 kDa apoprotein, molecular oxygen and coelenterazine (an imidazole compound with a molecular weight of 423, lilac pentagon). When aequorin is activated (second from the left) by Ca2+ (red sphere), it catalyzes the oxidation of coelenterazine to coelenteramide (yellow pentagon), which is in an excited state. Coelenteramide returns to its ground state (beige pentagon), emitting light at 470 nm, which is the blue light generated by purified aequorin. In vivo an energy transfer occurs from the excited state of the coelenteramide (complexed with aequorin and Ca2+) to GFP, which is responsible for the green fluorescence observed.

Some basic properties of A. victoria GFP

The evolutionary relationship of Aequorea GFP and Renilla GFP is not known, since the amino acid sequence of the latter has not yet been determined. The primary structure of Aequorea GFP was deduced from the cDNA sequence [9]. Aequorea GFP is a protein of 238 amino acids with a molecular weight of 27 or 30 kDa. There are several naturally occuring variants [3]. Native GFP is monomeric in dilute solutions (below 2 mg/mL) and dimeric in concentrated solutions (above 5 mg/mL) [3]. Whether dimeric GFP occurs in vivo is presently unknown [6].
GFP can be reversibly denatured to give a nonfluorescent protein [3]. Its spectral properties will be discussed in the chapter about the chromophore.
Futhermore, GFP is an exceptionally stable protein, as far as pH and temperature are concerned [3]. It is also quite resistant against proteolytic digestion [3]. The extraordinary stability is a consequence of its compact tertiary structure.

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Silke Jonda's PPS2 project
Structure and Function of GFP
updated 28.11.96