Interactive Tutorials - Spinning Disk Fundamentals

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Fluorescent Protein Technology

mOrange Fluorescent Protein Chromophore Formation

One of the most productive developments in the crusade to generate useful fluorescent proteins in the orange and red spectral regions resulted from the directed evolution of mRFP1. Nathan Shaner and Roger Tsien speculated that the chromophore amino acids, Gln66 and Tyr67, which are critical determinants of the spectral characteristics of the Aequorea proteins, would play a similar role in determining the color of mRFP1 derivatives as well. In this case, the directed evolution approach was applied to derivatives of mRFP1, targeting these amino acid residues followed by selecting for new color variants. The result was a group of six new monomeric fluorescent proteins exhibiting emission maxima ranging from 540 nanometers to 610 nanometers. These new fluorescent proteins were named mHoneydew, mBanana, mOrange, mTangerine, mStrawberry, and mCherry, referencing the common fruits that bear colors similar to their respective emission profiles. Thus, these new fluorescent proteins are commonly known as the mFruits. Although the mFruits were a tour de force, yielding tremendous information about fluorescent protein structure and function, several mFruit fluorescent proteins, including mHoneydew, mBanana, and mTangerine, suffer from low intrinsic brightness and poor photostability.

The most promising members in the mFruit series are mOrange, mStrawberry, and mCherry. The mOrange variant is the brightest of the mFruit proteins and has spectral characteristics allowing it to be paired with other fluorescent proteins in the cyan and green spectral region for multicolor imaging and as a potential FRET acceptor. Unfortunately, the photostability of mOrange is only approximately 5 percent that of EGFP. Recently, this deficiency was corrected by using a directed evolution approach that selected for enhanced photostability. This investigation yielded the mOrange2 derivative, which is about 30 percent more photostable than EGFP. mOrange2 also performs well as a fusion tag for cellular proteins, and the improved photostability should enable long-term imaging studies of cellular dynamics. The most useful red mFruit proteins, mCherry (610 nanometer emission peak) and mStrawberry (596 nanometer emission peak), have intrinsic brightness levels of approximately 50 and 75 percent that of EGFP, respectively. However, mCherry is more photostable than mStrawberry, so it is the preferred choice for cellular imaging. Recently, another red fluorescent protein, mApple, was generated in the same photostability screen that yielded mOrange2, and it features spectral characteristics close to mStrawberry, but with significantly improved photostability. The mApple variant is a rapidly maturing, bright, and photostable RFP that might prove to be better choice for tagging cellular proteins that are difficult to label (such as histones, tubulin, and connexins).

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 monomer 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.