Journal of Molecular Biology
Volume 401, Issue 5, 3 September 2010, Pages 776-791
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A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

https://doi.org/10.1016/j.jmb.2010.06.056Get rights and content

Abstract

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.

Introduction

In recent years, a handful of Aequorea green fluorescent protein (GFP) homologues cloned from anthozoan organisms have been reported to undergo irreversible photoconversion from a green fluorescent species to a red fluorescent species upon illumination with light of approximately 400 nm. To date, the naturally occurring photoconvertible proteins that have received the most attention are Kaede from coral Trachyphyllia geoffroyi,1 EosFP from stony coral Lobophyllia hemprichii,2 and Dendra from octocoral Dendronephthya sp.3 It has also been demonstrated that a naturally occurring nonphotoconvertible fluorescent protein (FP) can be engineered into a photoconvertible FP. Specifically, the photoconvertible FP known as KikGR was engineered from the green fluorescent variant KikG of the coral Favia favus.4

All green-to-red photoconvertible FPs characterized to date share a common His-Tyr-Gly-derived chromophore structure and a common photoconversion mechanism (Fig. 1).5 The newly synthesized protein first folds into the characteristic β-barrel structure of the Aequorea GFP superfamily6 and then undergoes the steps of posttranslational modification that lead to the formation of a green fluorescent chromophore with a conjugated system identical with that of Aequorea GFP chromophore. The chromophore can exist either in its neutral phenol form or in its anionic phenolate form (Fig. 1). Exactly where the equilibrium between these two forms lies is dependent on the local microenvironment of the chromophore (as determined by amino acid substitutions in close proximity to it) and the pH of the solution. The green-to-red photoconvertible FPs are distinguished from their nonphotoconvertible brethren by the respective consequences of exciting the neutral form, which absorbs most strongly at ∼ 400 nm. In wild-type Aequorea GFP, the excited state of the neutral species undergoes excited-state proton transfer to form the anionic species, which then emits green fluorescence.7 In the case of the green-to-red photoconvertible FPs, excitation of the neutral form leads to a break of the polypeptide chain through effective β-elimination of the residue immediately preceding the chromophore-forming His-Tyr-Gly tripeptide.5, 8 This elimination reaction results in the installation of a new double bond between Cα and Cβ of the His residue, placing the side-chain imidazole in conjugation with the remainder of the avGFP-type chromophore. This extended conjugation decreases the energy gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital, and shifts the emission to the orange-to-red region of the visible spectrum. Photoconversion (i.e., a light-induced change in excitation or emission wavelength maxima) via alternate mechanisms has been observed in other color classes of FP.9, 10

FP photoconversion allows researchers to ‘highlight’ a subpopulation of proteins within a cell or tissue through spatially defined illumination with a specific wavelength of light.11 The subsequent dynamics of the highlighted protein can be followed due to its distinct fluorescence excitation and emission.12, 13 Optical highlighting can also be achieved using FPs that undergo so-called photoactivation (irreversible conversion from a nonfluorescent species to a fluorescent species upon illumination)14 and photoswitching (reversible conversion between a nonfluorescent species and a fluorescent species upon illumination).15 A more recent application of these types of proteins that has generated excitement in the cell biology community is superresolution fluorescence imaging.16 This application also involves highlighting a subpopulation of protein molecules; however, instead of being in a spatially defined location, they are sparsely distributed throughout the whole sample to be imaged. The sample is then imaged to reveal the precise locations of the point sources of fluorescence, each of which corresponds to a single molecule of an FP. The sample is bleached, and the process is repeated. Many repetitions of this imaging protocol can produce images in which structures with dimensions of ∼ 50 nm can be resolved.17

Kaede, the first example of an FP that can undergo irreversible green-to-red photoconversion upon illumination with UV light, was initially described by Ando et al. in 2002.1 Unfortunately, the range of potential applications for Kaede remains limited by the fact that it is an obligate tetramer,18 and no monomeric variants have been reported. Unlike monomeric FPs, tetrameric FPs are generally detrimental to the proper trafficking and localization of recombinant fusion proteins.19 In producing a monomeric green-to-red photoconvertible FP, the same workers appear to have had more success with engineered variants of the tetrameric KikGR FP, which is substantially brighter and more efficiently photoconverted than Kaede.4 A monomeric version of KikGR, known as mKikGR, has recently been reported.20

The two other green-to-red photoconvertible FPs, EosFP and Dendra,2, 3 have been subjected to protein engineering to convert wild-type tetramers into monomers.17, 21 However, it is apparent that the monomeric version of EosFP retains a tendency to form dimers at higher concentrations.17 The monomeric variant of EosFP, known as mEos, was created through the introduction of two point mutations that disrupted the protein–protein interfaces of the tetrameric species.2, 22 Expression of mEos at temperatures greater than 30 °C is problematic,2 but an effectively monomeric tandem dimer variant does express well at 37 °C.23 The poor expression of mEos at 37 °C has been overcome with the engineering of mEos2 through targeted substitution of residues with solvent-exposed side chains.17 Although mEos2 has been reported to retain some propensity for dimer formation, this property does not appear to have adverse effects on the subcellular targeting of a variety of fusion proteins.17

The growing number of reports on optimized photoconvertible FPs reflects the growing demands on these proteins with respect to their enabling role in some popular cell biology applications. In particular, there is a continued need for bright and monomeric photoconvertible proteins, since these tools can enable the highest-precision superresolution fluorescence imaging.17, 20 In an effort to create a new monomeric photoconvertible FP with favorable properties, we embarked on a strategy distinct from that previously employed. Rather than start with a tetrameric photoconvertible protein and engineer it to be monomeric, we sought to start with a well-characterized monomeric coral-derived FP and engineer it to be a green-to-red photoconvertible FP. That tetrameric KikG was converted into KikGR through the use of protein design sets a precedent that it should be possible to convert a nonphotoconvertible FP into a photoconvertible FP.4 Our starting template is a monomeric version of Clavularia sp. cyan FP (cFP484, a wild-type Clavularia cyan FP) known as mTFP1 (monomeric teal FP 1).24

Section snippets

Guided consensus design of a new photoconvertible FP

The new photoconvertible FP based on the mTFP1 template was designed using a strategy analogous to that previously used to design a consensus FP based on a monomeric Azami green template.25 The known photoconvertible FPs, including EosFP,1 Dendra2,2 KikGR,4 and Kaede,3 were aligned to find the consensus at each amino acid position (Fig. 2). Amino acids of > 50% consensus were maintained in the designed protein. At positions with no clear consensus, the corresponding residue in mTFP1 was used.24

Summary

We have developed and characterized a new green-to-red photoconvertible FP variant known as mClavGR2. Unlike other members of this class of FP, mClavGR2 was engineered from a well-characterized and monomeric FP progenitor. Our results demonstrate that mClavGR2 has favorable spectral properties, retains the monomeric structure of its progenitor, and, in the constructs tested to date, does not interfere with the correct localization of a genetically fused protein partner. We anticipate this new

General methods and materials

All synthetic DNA oligonucleotides for cloning and library construction were purchased from Integrated DNA Technologies (Coralville, IA). PCR products and products of restriction digests were purified using the QIA gel extraction kit (Qiagen) in accordance with the manufacturer's protocols. Restriction enzymes were purchased from New England Biolabs. The cDNA sequences for all mClavGR variants were confirmed by dye terminator cycle sequencing using the DYEnamic ET kit (Amersham Biosciences) or

Competing Financial Interests Statement

New Fps that originate from the Campbell lab and are described in this manuscript are covered by a US patent application owned by the University of Alberta. Allele Biotechnology is the Licensed distributor of plasmids containing genes encoding these FPs.

Acknowledgements

This work was supported by Natural Sciences and Engineering Research Council of Canada Discovery grants (R.E.C. and C.W.C.) and a PetroCanada Young Innovator award to R.E.C. R.E.C. holds a Tier II Canada Research Chair in Bioanalytical Chemistry. The authors thank Ray Lemieux, Eric Flaim, Xuejun Sun, and Andreas Ibraheem at the University of Alberta for technical assistance, and Korey Wilson, Ericka Ramko, and Christopher Murphy at Florida State University for help with vector construction and

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    Competing financial interests statement: New fluorescent proteins that originate from the Campbell laboratory and are described in this work are covered by a US patent application owned by the University of Alberta. Allele Biotechnology is the licensed distributor of plasmids containing genes encoding these fluorescent proteins.

    1

    Present address: N. C. Shaner, Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA.

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