Orange fluorescent proteins (FPs) are attractive candidates as Förster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.
The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET)
microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Currently,
the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs.
However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the
characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has
advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we
characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET
standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and
acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the
fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting
from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and
lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red
FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this
observation.
Fluorescent proteins are the most common and versatile class of genetically encoded optical probes. While structure-guided
rational design and directed evolution approaches have largely overcome early problems such as oligomerization,
poor folding at physiological temperatures, and availability of wavelengths suitable for multi-color imaging, nearly all
fluorescent proteins have yet to be fully optimized. We have developed novel methods for evaluating the current
generation of fluorescent proteins and improving their remaining suboptimal properties. Little is yet known about the
mechanisms responsible for photobleaching of fluorescent proteins, and inadequate photostability is a chief complaint
among end users. In order to compare the performance of fluorescent proteins across the visual spectrum, we have
standardized a method used to measure photostability in live cells under both widefield and confocal laser illumination.
This method has allowed us to evaluate a large number of commonly used fluorescent proteins, and has uncovered
surprisingly complex and unpredictable behaviors in many of these proteins. We have also developed novel methods for
selecting explicitly for high photostability during the directed evolution process, leading to the development of highly
improved monomeric orange and red fluorescent proteins. These proteins, most notably our photostable derivative of
TagRFP, have remarkably high photostability and have proven useful as fusion tags for long-term imaging. Our methods
should be applicable to any of the large number of fluorescent proteins still in need of improved photostability.
KEYWORDS: Fluorescent proteins, Fluorescence resonance energy transfer, Proteins, Biosensors, Luminescence, Green fluorescent protein, Molecules, Sensors, Calcium, Resonance energy transfer
One of the most promising imaging techniques for monitoring dynamic protein interactions in living cells with optical
microscopy, universally referred to as FRET, employs the non-radiative transfer of energy between two closely adjacent
spectrally active molecules, often fluorescent proteins. The use of FRET in cell biology has expanded to such a degree
that hundreds of papers are now published each year using biosensors to monitor a wide spectrum of intracellular
processes. Most of these sensors sandwich an environmentally active peptide between cyan and yellow fluorescent
protein (CFP and YFP) derivatives to assay variables such as pH, calcium ion concentration, enzyme activity, or
membrane potential. The availability of these sensitive indicators is growing rapidly, but many are hampered by a low
dynamic range that often is only marginally detectable over the system noise. Furthermore, extended periods of
excitation at wavelengths below 500 nm have the potential to induce phototoxic effects that can mask or alter the
biological events under observation. Recent success in expanding the fluorescent protein color palette offers the
opportunity to explore new FRET partners that may be suitable for use in advanced biosensors.
Despite the explosive growth of the Internet (in terms of the World Wide Web) as an informational resource for the original scientific literature pertaining to fluorescent protein investigations, there remains an obvious void in educational Websites targeted at beginning students and novices in the field. To address this issue, educational sites dedicated to optical microscopy and digital imaging being constructed and hosted at The Florida State University are turning their attention to the increasing application of fluorescent proteins for live-cell imaging studies. The primary focus of this effort is to create new sections of the sites that address the structure and properties of fluorescent proteins as well as optimizing their utility in imaging experiments.
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