Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Fluorescent Protein Technology

mKusabira Orange Fluorescent Protein Chromophore Formation

In the past several years, a relatively large number of potentially useful orange fluorescent proteins have emerged from various Anthozoa species. In one of the first examples, a protein named Kusabira Orange (KO) was isolated from the mushroom coral Fungia concinna (known in Japanese as Kusabira-Ishi). The sequence encoding KO was engineered to add ten amino acids to the N-terminus, resulting in a fluorescent protein having an absorption maximum at 548 nanometers (ideal for excitation with a 543-nanometer laser) while emitting bright orange fluorescence at 561 nanometers. In an effort similar to the strategy used to generate mRFP1, a monomeric version of Kusabira Orange (mKO) was created after site-directed and random mutagenesis of 20 amino acids.

The monomeric Kusabira orange exhibits similar spectral properties to the tetramer and has a brightness value similar to EGFP, but is slightly more sensitive to acidic environments. However, the photostability of this fluorescent protein under arc lamp illumination is exceptional, making mKO an excellent choice for long-term imaging experiments. Furthermore, the emission spectral profile is sufficiently well separated from cyan fluorescent proteins to increase the FRET efficiency in biosensors incorporating mKO, and the probe is useful in multicolor investigations with a combination of cyan, green, yellow, and red fluorescent proteins. Recently, a fast-folding variant containing 8 additional mutations, named mKO2, was developed and should improve the utility of this probe for live cell imaging.

During protein maturation, both chromophores undergo a second oxidation step to produce an acylimine linkage in the polypeptide backbone (typical of red Anthozoa fluorescent proteins) followed by the spontaneous formation of a novel third ring system. In the case of mOrange, this third ring is an oxazole formed by the threonine residue at position 66, whereas in mKusabira Orange, the ring is a thiazole formed by the cysteine residue at position 65. Thus, these orange fluorescent proteins have emission profiles that are blue-shifted (from red to shorter orange wavelengths) relative to the red variants due to elimination of the conjugation between the acylimine carbonyl and the chromophore. This phenomenon is similar to the yellow emission arising from reduced conjugation observed in ZsYellow when the lysine residue at position 66 cyclizes with the acylimine to form a partially unsaturated piperidine ring system.

Contributing Authors

Tony B. Gines, Kevin A. John, Tadja Dragoo and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.