GFP-like proteins: identification, analysis and applications

Lukyanov K, Fradkov A, Gurskaya N, Yanushevich Yu, Chudakov D, Bulina M, Yampolsky I, Mudrik N.
Collaborations: CLONTECH Laboratories, Inc.
Mikhail V. Matz, Whitney Laboratory University of Florida
Vladislav V. Verkhusha, Albert Einstein College of Medicine, Bronx, NY, USA
T.W.J. Gadella, University of Amsterdam, Amsterdam, Netherlands
Carsten Schultz, EMBL, Heidelberg, Germany
Evrogen JSC

Green fluorescent protein (GFP) was discovered in hydroid medusa Aequorea victoria (synonyms A. forskalea, A. aequorea) more than 40 years ago. After that, GFPs were found in several bioluminescent Hydrozoa and Anthozoa species. In all these examples GFPs played role of secondary emitter within bioluminescent systems. The association of GFPs with bioluminescence was probably the main reason why researchers did not search for GFP-like proteins in non-bioluminescent corals for a long time.
We were lucky to clone genes for GFP-like proteins from non-bioluminescent Anthozoa for the first time. Fortune really smiled on us: in just a few months after the work was started we cloned the first GFP from the sea anemone Anemonia majano. In comparison to many other marine coelenterates, working on reef Anthozoa is particularly convenient since one can readily buy the specimens for several dollars in aquarium shops throughout the world. We did not organize expeditions to collect our first samples, but found brightly fluorescent and colored sea anemones and corallimorphs (mushroom anemones) in Moscow instead. Shortly after the publication of our paper, other groups reported cloning of GFP-like proteins from Anthozoa species. At present, it is widely accepted that vivid fluorescent and non-fluorescent coloration of reef Anthozoa is mainly determined by GFP homologs.
Discovery of GFP-like proteins in Anthozoa species led to significant expansion of our knowledge about this amazing protein family. The most striking feature of the novel proteins is a very broad diversity of their spectral properties. During the past four years, considerable progress was achieved in the world in both understanding structure of Anthozoa fluorescent proteins (FPs) and their improvement for in vivo labeling uses.

• Discovery of GFP-like fluorescent proteins of different colors (including first red FP) in Anthozoa species (Matz et al., 1999).
• Discovery of GFP-like non-fluorescent chromoproteins (Lukyanov et al., 2000).
• Generation of far-red fluorescent proteins on the base of non-fluorescent chromoproteins (Gurskaya et al., 2001b; Shkrob et al., 2005).
• Investigation of color transitions in GFP-like proteins by mutagenesis (Gurskaya et al., 2001a; Bulina et al., 2002).
• Generation of non-aggregating mutants of Anthozoa FPs (Yanushevich et al., 2002).
• Creation of a palette of so called photoactivatable fluorescent proteins: Kindling Fluorescent Proteins (KFPs; Chudakov et al., 2003a; Chudakov et al., 2003b), PS-CFP (Chudakov et al., 2004), Dendra (Gurskaya et al., 2006).
• Revealing an unexpected color diversity of GFP-like proteins in Hydrozoa species (Shagin et al., 2004).
• Discovery of GFP-like proteins in crustaceans (Shagin et al., 2004).
• Development of the first genetically encoded photosensitizer - phototoxic fluorescent protein KillerRed (Bulina et al., 2006a, 2006b).
• Construction of HyPer - the first genetically encoded sensor for hydrogen peroxide (Belousov et al., 2006).

Characterization and engineering of GFP homologs is very far from completion. Extensive crystallographic, biochemical and biophysical studies should be performed to approach adequate understanding of structure-function relationships in these proteins that would ultimately make it possible to engineer FP variants of any desirable properties. Evolutionary diversity of GFP-like proteins in animal kingdom should be investigated. For practical biotechnology needs, fluorescent probes of the following kind appear to be in especially high demand. First, far-red FPs should be either found among natural FPs or created on the basis of chromoproteins with absorption in far-red region. This direction is especially important for imaging on the level of whole animals since far-red light penetrates animal tissues most easily. Second, a new generation of sensitive and highly contrast fluorescent molecular sensors should be engineered for real-time monitoring of changes in intracellular concentration of ions, radicals, secondary messengers and other key events. Also, several monomeric photoactivatable tags of distinct colors and wavebands of activating light should be developed for simultaneous tracking different target proteins.

This work is supported by grants from:

• Russian Academy of Sciences, program "Molecular and Cell Biology".
• EU Framework Program 6 Integrated Project "Integrated Technologies for In-Vivo Molecular Imaging", contract LSHG-CT-2003-503259. www.molimg.gr
• Howard Hughes Medical Institute grant HHMI 55005618 "Genetically encoded photosensitizers for light-induced cell killing and protein inactivation".
• National Institute of Health grant 5 R01-GM070358-04 "Kindling fluorescent proteins for tagging and biosensors".