Interactive Tutorials - Spinning Disk Fundamentals

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Fluorescent Protein Technology

DsRed Fluorescent Protein Chromophore Formation

One of the first Anthozoa-derived fluorescent proteins to be extensively characterized was isolated from the sea anemone Discosoma striata. This novel fluorescent protein was originally called drFP583, but is now commonly known as DsRed. When expressed in cells, the fully matured DsRed fluorescent protein is optimally excited at 558 nanometers, and has an emission maximum at 583 nanometers. However, there are multiple problems associated with DsRed when used for live cell imaging. The maturation of DsRed is slow, and proceeds through an intermediate chromophore stage where most of the fluorescence emission occurs in the green spectral region. This "green state" introduces signal crosstalk that limits the utility of DsRed for multiple labeling experiments. In addition, DsRed is an obligate tetramer with the tendency to form oligomers, which can lead to protein aggregation and interference with the localization of protein fusions in living cells. Although these side effects are not important when the probe is utilized simply as a reporter for gene expression, the application of DsRed for a wide variety of investigations in cell biology is severely compromised.

Several of the major problems associated with DsRed were overcome by site-directed and random mutagenesis approaches. This effort yielded a second-generation version of DsRed, appropriately called DsRed2, which contains a series of silent nucleotide substitutions corresponding to human codon preferences, as well as several mutations that increase the maturation rate. In addition, the elimination of a string of basic amino acid residues at the amino terminus of DsRed2 (by mutation to acidic or neutral moieties) significantly reduces the tendency of the protein to form aggregates. DsRed2 still forms a tetramer in solution, but the increased maturation rate greatly reduces the level of the intermediate green species, making it is more useful for multiple labeling experiments. Further enhancement in the rate of maturation was realized with the third generation of DsRed mutants, which also display an increased intrinsic brightness. For example, the DsRed-Express variant (available from Clontech) can be detected within an hour after transfection of cells, compared to approximately six hours for DsRed2 and 11 to 15 hours for DsRed. More advanced versions of DsRed, named DsRed-Express2 and DsRed-Max, exhibit faster maturation rates and improved solubility. However, since these direct descendents of DsRed remain obligate tetramers, there has been a concerted effort to generate dimeric and monomeric red fluorescent protein variants.

The generation of truly monomeric DsRed variants, as well as monomers from proteins derived from a host of different Anthozoa species, has proven to be a difficult task. For example, site-directed mutagenesis to break the tetramer formation by DsRed2 resulted in the generation of a non-fluorescent monomer. To rescue fluorescence, investigators applied successive rounds of random mutagenesis to the monomer, selecting for proteins in each round with improved red fluorescence (this approach is called directed evolution). A total of 33 amino acid substitutions were required to generate the first-generation monomeric red fluorescent protein, which was termed mRFP1. The rapidly maturing mRFP1 overcame many critical problems associated with DsRed, while shifting the fluorescence emission about 25 nanometers deeper into the red spectrum. Unfortunately, mRFP1 exhibits significantly reduced fluorescence emission intensity (as expected, the quantum yield of the monomer is about 25 percent of DsRed2) and it is very sensitive to photobleaching. Furthermore, mRFP1 has an absorbance peak at 503 nanometers that arises from a non-fluorescent species, which indicates that a significant fraction of the protein never fully matures. Over the past several years, extensive mutagenesis efforts, including novel techniques such as iterative somatic hypermutation, have successfully been applied to mRFP1 to yield a new generation of orange, red, and far-red fluorescent protein variants.

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.

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.

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