Andrew B. Cubitt

Crystal structure of the Aequorea victoria green fluorescent protein.

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded β barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

Understanding, improving and using green fluorescent proteins

Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.

On/off blinking and switching behaviour of single molecules of green fluorescent protein.

Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission (‘blinking’) on a timescale of several seconds—behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.

UNDERSTANDING STRUCTURE-FUNCTION RELATIONSHIPS IN THE AEQUOREA VICTORIA GREEN FLUORESCENT PROTEIN

Publisher Summary Learning the physiological role of green fluorescent protein (GFP) and its interaction with aequorin could help understand how to create mutants that exhibit efficiency energy transfer and how to effectively control dimerization. The interaction of GFP with aequorin is readily reversible and is stabilized by high protein and salt concentrations—conditions likely to be encountered within the light-emitting organelles of Aequorea victoria. Both GFP and aequorin can dimerize under appropriate conditions, and it is the dimerized forms that are believed to interact. The molecular details of the interaction of aequorin with GFP are unknown, although it has been suggested that a C-terminal hydrophobic patch, deriving from amino acids 206,221, and 223, or a stretch of negative electrostatic potential could be plausible interaction domains. Although the significance of the heterogeneity of both GFP and aequorin has largely been ignored, it is possible that it favors the correct association of GFP with aequorin. At least one site of heterogeneity in the Aequorea-derived GFP nucleotide residues occurs at a position involved in GFP Dimerization. Isoform variation at this position was split between positively and negatively charged amino acids, which would favor the selective association of different isoforms.