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Enter the name for this tabbed section: 57 Citation
57. H-w. Ai, M.A. Baird, Y. Shen, M.W. Davidson*, and R.E. Campbell*, “Engineering and characterizing monomeric fluorescent proteins for live cell imaging applications”. Nat. Protocols, 2014, Accepted. Jan. 3, 2014.
Enter the name for this tabbed section: 57 Abstract
Naturally occurring fluorescent proteins cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation, and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated using protein engineering and directed evolution, resulting in superior probes for use in live cell fluorescence microscopy. In this protocol we provide methods for: engineering a monomeric fluorescent protein; enhancing its brightness by directed evolution; and thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features such as a red-shifted emission spectrum or enhanced photostability. While the length of the procedure is dependent upon the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.
Enter the name for this tabbed section: 57 Full Text Options
Funding: University of Alberta, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council of Canada (Discovery grant), and Alberta Ingenuity (Scholarship to Y.S. and a New Faculty Award to R.E.C.).
Enter the name for this tabbed section: 56 Citation
56. A.S.F. Belal, B.R. Sell, H. Hoi, M.W. Davidson, and R.E. Campbell*, “Optimization of a Genetically Encoded Biosensor for Cyclin B1-Cyclin Dependent Kinase 1”. Mol. Biosyst., 2014, 10(2), 191-195.
Enter the name for this tabbed section: 56 Abstract
Fluorescent protein (FP)-based biosensors have revolutionized the ability of researchers to monitor enzyme activities in live cells. While the basic design principles for FP-based biosensors are well established, first-generation biosensor constructs typically suffer from relatively low fluorescence responses that limit their general applicability. The protein engineering efforts required to substantially improve the biosensor responses are often both labour and time intensive. Here we report the application of a high throughput bacterial colony screen for improving the response of kinase biosensors. This effort led to the development of a second-generation cyclin B1-CDK1 biosensor with a 4.5-fold greater response than the first-generation biosensor.
Enter the name for this tabbed section: 56 Full Text Options
Online article (open access)
Supplementary Material
PMID: 24281384
Funding: NSERC Discovery.
Enter the name for this tabbed section: 55 Citation
55. H. Hoi, Y. Ding, and R.E. Campbell*, “FRET with Fluorescent Proteins”, in FRET - Förster Resonance Energy Transfer: From Theory to Applications (Eds. Nikko Hildebrandt and Igor Medintz) Wiley, November 2013, pages 431-473.
Enter the name for this tabbed section: 55 Abstract
Fluorescent proteins (FPs) are a class of homologous proteins that share the remarkable ability to form a visible wavelength fluorophore from their own amino acid residues. The discovery and subsequent engineering and optimization of these proteins has provided biologists with a plethora of tools for the imaging of subcellular structures, protein dynamics, metabolite concentrations, and even enzyme activities in live cells. In recognition of the broad impact of these proteins, the Nobel Prize in Chemistry for 2008 was awarded to 3 of the pioneers who made key contributions to the early developments in this field.
In this chapter we aim to provide a survey of the development and use of fluorescent proteins in FRET sensing and other FRET based applications. In addition, we include in-depth discussion of several examples from the literature with particular emphasis on original developments that have occurred during the last two years prior to the time of writing (2008-2010). It should be noted that a vast number of reviews have been written on essentially every aspect of FPs during the last 15 years. We include references to only the most relevant reviews in each section of this chapter and have tried to avoid too much redundancy in our citing of review articles. We apologize in advance for any citations that the reader feels we have erroneously excluded.
In this chapter we aim to provide a survey of the development and use of fluorescent proteins in FRET sensing and other FRET based applications. In addition, we include in-depth discussion of several examples from the literature with particular emphasis on original developments that have occurred during the last two years prior to the time of writing (2008-2010). It should be noted that a vast number of reviews have been written on essentially every aspect of FPs during the last 15 years. We include references to only the most relevant reviews in each section of this chapter and have tried to avoid too much redundancy in our citing of review articles. We apologize in advance for any citations that the reader feels we have erroneously excluded.
Enter the name for this tabbed section: 55 Full Text Options
Online book
Link to online chapter
Published Online: 4 OCT 2013
DOI: 10.1002/9783527656028.ch11
ISBN: 978-3-527-32816-1.
Chapter: 42 pages, 185 references.
Book: 816 pages.
Funding: NSERC Discovery and CIHR NHG 99085.
Enter the name for this tabbed section: 54 Citation
54. H. Hoi, E.S. Howe, Y. Ding, W. Zhang, M.A. Baird, B.R. Sell, J.R. Allen, M.W. Davidson, and R.E. Campbell*, “An engineered monomeric Zoanthus sp. yellow fluorescent protein”, Chem. Biol., 2013, 20, 1296-1304.
Enter the name for this tabbed section: 54 Abstract
Protein engineering has created a palette of monomeric fluorescent proteins (FPs), but there remains a ~30 nm spectral gap between the most red-shifted useful Aequorea victoria green FP (GFP) variants and the most blue-shifted useful Discosoma sp. red FP (RFP) variants. To fill this gap, we have engineered a monomeric version of the yellow FP (YFP) from Zoanthus sp. coral. Our preferred variant, designated as mPapaya1, displays excellent fluorescent brightness, good photostability, and retains its monomeric character both in vitro and in living cells in the context of protein chimeras. We demonstrate that mPapaya1 can serve as a good Förster resonance energy transfer (FRET) acceptor when paired with an mTFP1 donor. mPapaya1 is a valuable addition to the palette of FP variants that are useful for multicolor imaging and FRET-based biosensing.
Enter the name for this tabbed section: 54 Full Text Options
Online Article
Journal highlight by Whittredge and Taraska
Supporting Information
Funding: NSERC Discovery and Alberta Innovates Technology Futures (AITF) Scholarship to W.Z.
Enter the name for this tabbed section: 53 Citation
53. H.J. Carlson and R.E. Campbell*, “Mutational analysis of a red fluorescent protein-based calcium ion indicator”, Sensors, 2013, 13, 11507-11521.
Enter the name for this tabbed section: 53 Abstract
As part of an ongoing effort to develop genetically encoded calcium ion (Ca2+) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds to Ca2+-bound CaM. In contrast to the closely related Ca2+ indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca2+ and a sensing mechanism that does not involve direct modulation of the chromophore pKa. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca2+ binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the Kd for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.
Enter the name for this tabbed section: 53 Full Text Options
Special Issue "Fluorescent Biosensors"
Open Access article
Supplementary material
DOI:10.3390/s130911507
Received: 11 August 2013; in revised form: 27 August 2013 / Accepted: 29 August 2013 / Published: 2 September 2013
Funding: NSERC Discovery, NSERC PGSM, and Alberta Ingenuity Scholarship
Enter the name for this tabbed section: 52 Citation
52. H.J. Carlson and R.E. Campbell*, “Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry”, Protein Eng. Des. Sel., 2013, 26, 763-772.
Enter the name for this tabbed section: 52 Abstract
Red fluorescent indicators for calcium ion (Ca2+) are preferable, relative to blue-shifted alternatives, for biological imaging applications due to the lower phototoxicity, lower autofluorescent background and deeper tissue penetration associated with longer wavelength light. Accordingly, we undertook the development of a genetically encoded Ca2+ indicator based on the popular and widely utilized Discosoma-derived red fluorescent protein, mCherry. Starting from a promising but dimly fluorescent circular permutated variant of mCherry, we first engineered a 13-fold brighter variant (cp196V1.2) through directed evolution. This bright cp196V1.2 was then used as the scaffold for creation of eight distinct libraries of potential Ca2+ indicators via permutation at different sites within the 7th and 10th β-strands, and fusion of calmodulin and M13 to the new termini. Screening of these libraries led to the conclusion that, consistent with previous investigations of homologous fluorescent proteins, the 146–145 site in β-strand 7 is the most promising permutation site for construction of useful Ca2+ indicators. Further rounds of directed evolution ultimately led to an indicator that exhibits a 250% change in intrinsic brightness in response to Ca2+ and an exceptionally high affinity (Kd = 6 nM) for Ca2+.
Enter the name for this tabbed section: 52 Full Text Options
Journal abstract
Supplementary material
PMID: 24151339
First published online: October 22, 2013
DOI: 10.1093/protein/gzt052
Funding: NSERC Discovery, NSERC PGSM, and Alberta Ingenuity Scholarship
Enter the name for this tabbed section: 51 Citation
51. E.M. Lynes, A. Raturi, M. Shenkman, C.O. Sandova, M.C. Yap, J. Wu, A. Janowicz, N. Myhill, M.D. Benson, R.E. Campbell, L. G. Berthiaume, G.Z. Lederkremer and T. Simmen*, “Palmitoylation is the Switch that Assigns Calnexin to Quality Control or ER Calcium Signaling”, J. Cell Sci., 2013, 126, 3893-3903.
Enter the name for this tabbed section: 51 Abstract
The palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) Ca2+ transport ATPase (SERCA) 2b and that this interaction determines ER Ca2+ content and the regulation of ER–mitochondria Ca2+ crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER-stress-dependent mechanism. Following a short-term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of Ca2+ signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions, or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to Ca2+ signaling or to protein chaperoning.
Enter the name for this tabbed section: 51 Full Text Options
Journal abstract
Supplementary material
DOI: 10.1242/jcs.125856
Funding: CIHR NHG 99085
Enter the name for this tabbed section: 50 Citation
50. J. Wu, L. Liu, T. Matsuda, Y. Zhao, A. Rebane, M. Drobizhev, Y-F. Chang, S. Araki, Y. Arai, K. March, T.E. Hughes, K. Sagou, T. Miyata, T. Nagai*, W-H. Li*, R.E. Campbell*, “Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications”, ACS Chem. Neurosci., 2013, 4, 963–972
Enter the name for this tabbed section: 50 Abstract
We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.
Enter the name for this tabbed section: 50 Full Text Options
Journal Abstract
Supporting Information
Highlighted at Openoptogenetics.org
Supporting Information
Highlighted at Openoptogenetics.org
Publication Date (Web): March 1, 2013.
DOI: 10.1021/cn400012b
Funding: CIHR NHG 99085, CIHR MOP 123514, NSERC Discovery, and Alberta Ingenuity Nanotechnology Scholarship to Y.Z.
Enter the name for this tabbed section: 49 Citation
49. H. Hoi, T. Matsuda, T. Nagai, and R.E. Campbell*, “Highlightable Ca2+ indicators for live cell imaging”, J. Am. Chem. Soc., 2013, 135, 46-49.
Enter the name for this tabbed section: 49 Abstract
Two of the most powerful implementations of fluorescent protein (FP) technology are “highlighters”, which can be converted from nonfluorescent to fluorescent or from one color to another by illumination, and calcium ion (Ca2+) indicators. Combining the properties of both of these FP classes into a single construct would produce a highlightable Ca2+ indicator that would enable researchers to mark a single cell spectrally in a transfected tissue and image its intracellular Ca2+ dynamics. In an effort to create such a hybrid tool, we explored three different protein design strategies. The strategy that ultimately proved successful involved the creation of a circularly permuted version of a green-to-red photoconvertible FP and its introduction into a G-CaMP-type single-FP-based Ca2+ indicator. Optimization by directed evolution led to the identification of two promising variants that exhibit excellent photoconversion properties and have an up to 4.6-fold increase in red fluorescence intensity upon binding of Ca2+. We demonstrate the utility of these variants in HeLa cells and rat hippocampal neurons.
Enter the name for this tabbed section: 49 Full Text Options
Journal Abstract
Supporting Information
Highlighted at Openoptogenetics.org
Accepted Dec. 20, 2012.
DOI: 10.1021/ja310184a.
Funding: NSERC Discovery
Supporting Information
Highlighted at Openoptogenetics.org
Accepted Dec. 20, 2012.
DOI: 10.1021/ja310184a.
Funding: NSERC Discovery
Enter the name for this tabbed section: 48 Citation
48. S.C. Alford, J. Wu, Y. Zhao, R.E. Campbell, and T. Knöpfel*, “Optogenetic Reporters”. Biol. Cell, 2013, 105, 14-29.
Enter the name for this tabbed section: 48 Abstract
The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live-cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.
Enter the name for this tabbed section: 48 Full Text Options
Journal article
DOI: 10.1111/boc.201200054
Accepted Oct. 30, 2012.
Accepted manuscript online: Nov. 6, 2012.
Highlighted at ChemistryViews
Funding: CIHR NHG 99085, NSERC Discovery, NSERC CGSD3 to S.C.A., Alberta Ingenuity Ph.D. Scholarship to S.C.A., and Alberta Ingenuity Nanotechnology Scholarship to Y.Z.
Enter the name for this tabbed section: 47 Citation
47. A.L. McEvoy*, H. Hoi, M. Bates, E. Platonova, P.J. Cranfill, M.W. Davidson, H. Ewers, J. Liphardt, and R.E. Campbell*, “mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities”. PLoS ONE, 2012, 7(12): e51314.
Enter the name for this tabbed section: 47 Abstract
Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.
Enter the name for this tabbed section: 47 Full Text Options
DOI:10.1371/journal.pone.0051314
Accepted Oct. 31, 2012.
Funding: NSERC Discovery
Enter the name for this tabbed section: 46 Citation
46. S.C. Alford, Y. Ding, T. Simmen, and R.E. Campbell*, “Dimerization-Dependent Green and Yellow Fluorescent Proteins”. ACS Synth. Biol., 2012, 1, 569-575.
Enter the name for this tabbed section: 46 Abstract
Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pKa for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.
Enter the name for this tabbed section: 46 Full Text Options
Journal Abstract
Supporting Information
High Resolution cover
Author Feature
DOI: 10.1021/sb300050j
Publication Date (Web): August 9, 2012
Funding: CIHR NHG 99085, NSERC Discovery, NSERC CGSD3 to S.C.A., and Alberta Ingenuity Ph.D. Scholarship to S.C.A.
Supporting Information
High Resolution cover
Author Feature
DOI: 10.1021/sb300050j
Publication Date (Web): August 9, 2012
Funding: CIHR NHG 99085, NSERC Discovery, NSERC CGSD3 to S.C.A., and Alberta Ingenuity Ph.D. Scholarship to S.C.A.
Enter the name for this tabbed section: 45 Citation
45. M. Funes-Huacca, A. Wu, E. Szepesvari, P. Rajendran, N. Kwan-Wong, A. Razgulin, Y. Shen, J. Kagira, R.E. Campbell and R. Derda*, “Portable self-contained cultures for phage and bacteria made of paper and tape”. Lab Chip, 2012, 12, 4269-4278.
Enter the name for this tabbed section: 45 Abstract
In this paper, we demonstrate that a functional, portable device for the growth of bacteria or amplification of bacteriophage can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen but non-permeable to water. These devices supply bacteria with oxygen and prevent the evaporation of media for a period over 48 h. The division time of E. coli and the amplification of the phage M13 in this device are similar to the rates measured on agar plates and in shaking cultures. The growth of bacteria with a fluorescent mCherry reporter can be quantified using a flatbed scanner or a cell phone camera. Permeating devices with commercial viability dye (PrestoBlue) can be used to detect low copy number of E. coli (1–10 CFU in 100 μL) and visualize microorganisms in environmental samples. The platform, equipped with bacteria that carry inducible mCherry reporter could also be used to quantify the concentration of the inducer (here, arabinose). Identical culture platforms can, potentially, be used to quantify the induction of gene expression by an engineered phage or by synthetic transcriptional regulators that respond to clinically relevant molecules. The majority of measurement and fabrication procedures presented in this report have been replicated by low-skilled personnel (high-school students) in a low-resource environment (high-school classroom). The fabrication and performance of the device have also been tested in a low-resource laboratory setting by researchers in Nairobi, Kenya. Accordingly, this platform can be used as both an educational tool and as a diagnostic tool in low-resource environments worldwide.
Enter the name for this tabbed section: 45 Full Text Options
Journal Abstract
DOI: 10.1039/C2LC40391A
Accepted: 10 Jul 2012
First published on the web: 15 Aug 2012
Funding: NSERC Discovery and Alberta Ingenuity Nanotechnology Scholarship to Y.S.
DOI: 10.1039/C2LC40391A
Accepted: 10 Jul 2012
First published on the web: 15 Aug 2012
Funding: NSERC Discovery and Alberta Ingenuity Nanotechnology Scholarship to Y.S.
Enter the name for this tabbed section: 44 Citation
44. P. Tewson, M. Westenberg, Y. Zhao, R.E. Campbell, A.M. Quinn, T.E. Hughes,* “Simultaneous detection of Ca2+ and diacylglycerol signaling in living cells”. PLoS ONE, 2012, 7(8): e42791.
Enter the name for this tabbed section: 44 Abstract
Phospholipase C produces two second messengers - diacylglycerol (DAG), which remains in the membrane, and inositol triphosphate (IP3), which triggers the release of calcium ions (Ca2+) from intracellular stores. Genetically encoded sensors based on a single circularly permuted fluorescent protein (FP) are robust tools for studying intracellular Ca2+ dynamics. We have developed a robust sensor for DAG based on a circularly permuted green FP that can be co-imaged with the red fluorescent Ca2+ sensor R-GECO for simultaneous measurement of both second messengers.
Enter the name for this tabbed section: 44 Full Text Options
Open access article
PDF version
Funding: CIHR NHG 99085 and Alberta Ingenuity Nanotechnology Scholarship to Y.Z.
PDF version
Funding: CIHR NHG 99085 and Alberta Ingenuity Nanotechnology Scholarship to Y.Z.
Enter the name for this tabbed section: 43 Citation
43. R.E. Campbell*, “New Bioanalytical Tools and Devices: Chemistry leads the way”. Biotechnology Focus (Bioscienceworld), 2012, 16(4), 7-9.
Enter the name for this tabbed section: 43 Abstract
Three University of Alberta chemists are developing innovative and imaginative bioanalytical techniques aimed at tackling the burden and suffering caused by infectious diseases in the developing world.
Enter the name for this tabbed section: 43 Full Text Options
Online text version
Interactive PDF version
Highlighting the research of Drs. Gibbs-Davis, Serpe, and Derda
Enter the name for this tabbed section: 42 Citation
42. K.D. Daze, T. Pinter, C.S. Beshara, A. Ibraheem, S.A. Minaker, M.C.F. Ma, R.J.M. Courtemanche, R.E. Campbell, and F. Hof*, “Supramolecular hosts that recognize methyllysines and disrupt the interaction between a modified histone tail and its epigenetic reader protein”. Chem. Sci., 2012, 3, 2695-2699.
Enter the name for this tabbed section: 42 Abstract
Post-translational modifications of proteins (including phosphorylation, acetylation and methylation, among others) frequently carry out their biological functions by serving as ‘on’ switches for protein–protein interactions. As highly localized and perfectly defined hot-spots for protein–protein binding, they are a diverse set of motifs that collectively offer great promise as targets for therapeutic intervention and fundamental studies of chemical biology. Recent years have seen the discovery of a very large number of such modification sites on the unstructured tails of proteins, including histones and the tumor suppressor p53. These unstructured protein elements do not present concave binding pockets, and as such cannot be targeted by the conventional small-molecule agents of chemical biology and medicinal chemistry. We report here a family of calixarene-based supramolecular hosts that bind selectively and with high affinity to histone trimethyllysine motifs that are relevant to gene regulation and oncogenesis. We show that these compounds constitute a novel class of protein–protein interaction disruptors and that they can operate selectively against their targeted trimethyllysine sites even in highly complex protein substrates bearing a background of many unmethylated lysines and arginines.
Enter the name for this tabbed section: 42 Full Text Options
Enter the name for this tabbed section: 41 Citation
41. S.C. Alford, A.S. Abdelfattah, Y. Ding, R.E. Campbell*, "A Fluorogenic Red Fluorescent Protein Heterodimer". Chem. Biol., 2012, 19, 353-360.
Enter the name for this tabbed section: 41 Abstract
The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a Kd of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca2+-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.
Enter the name for this tabbed section: 41 Full Text Options
Journal Abstract
DOI: 10.1016/j.chembiol.2012.01.006
Research Highlight: Erika Pastrana, "Together we shine", Nature Methods 9, 432–433 (2012)
Funding: CIHR NHG 94487/99085, NSERC Discovery, NSERC CGSD3 to S.C.A., and Vanier CGS to A.S.A.
DOI: 10.1016/j.chembiol.2012.01.006
Research Highlight: Erika Pastrana, "Together we shine", Nature Methods 9, 432–433 (2012)
Funding: CIHR NHG 94487/99085, NSERC Discovery, NSERC CGSD3 to S.C.A., and Vanier CGS to A.S.A.
Enter the name for this tabbed section: 40 Citation
40. Y. Ding, H-w. Ai, H. Hoi, R.E. Campbell*, "FRET-based biosensors for multiparameter ratiometric imaging of Ca2+ dynamics and caspase-3 activity in single cells". Analytical Chemistry, 2011, 83, 9687–9693.
Enter the name for this tabbed section: 40 Abstract
As one of the principal cytoplasmic second messengers, the calcium ion (Ca2+) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca2+ in live cells using noninvasive methods such as genetically encoded biosensors based on Förster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca2+ biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca2+ affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca2+ in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.
Enter the name for this tabbed section: 40 Full Text Options
Journal Abstract
Link to PDF
DOI: 10.1021/ac202595g
Online : November 14, 2011
Funding: CIHR NHG 94487/99085 and NSERC Discovery
Link to PDF
DOI: 10.1021/ac202595g
Online : November 14, 2011
Funding: CIHR NHG 94487/99085 and NSERC Discovery
Enter the name for this tabbed section: 39 Citation
39. A. Ibraheem, H. Yap, Y. Ding, R.E. Campbell*, "A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors". BMC Biotechnology, 2011, 11, 105.
Enter the name for this tabbed section: 39 Abstract
Background
Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Forster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.
Results
To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.
Conclusion
Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3x improved relative to that of the initially constructed biosensor (29%).
Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Forster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.
Results
To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.
Conclusion
Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3x improved relative to that of the initially constructed biosensor (29%).
Enter the name for this tabbed section: 39 Full Text Options
Enter the name for this tabbed section: 38 Citation
38. Y. Zhao, S. Araki, J. Wu, T. Teramoto, Y-F. Chang, M. Nakano, A.S. Abdelfattah, M. Fujiwara, T. Ishihara, T. Nagai, and R.E. Campbell*, "An Expanded Palette of Genetically Encoded Ca2+ Indicators". Science, 2011, 333, 1888-1891.
Enter the name for this tabbed section: 38 Abstract
Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments; and, in conjunction with a CFP-YFP–based indicator, Ca2+ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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Journal Abstract
PDF reprint
Supplementary Material
Issue: 30 September 2011
Online: 8 September 2011
DOI:10.1126/science.1208592
PDF reprint
Supplementary Material
Issue: 30 September 2011
Online: 8 September 2011
DOI:10.1126/science.1208592
Funding: CIHR NHG 94487/99085, NSERC Discovery, Alberta Ingenuity Nanotechnology Scholarship to Y.Z., and Vanier CGS to A.S.A.
Enter the name for this tabbed section: 37 Citation
37. H. Hoi, N.C. Shaner, M.W. Davidson, C.W. Cairo, J. Wang, R.E. Campbell*, “A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization”. J. Mol. Biol., 2010, 401, 776-791.
Enter the name for this tabbed section: 37 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.
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Enter the name for this tabbed section: 36 Citation
36. H.J. Carlson, D. Cotton, and R.E. Campbell*, “Circularly permuted monomeric red fluorescent proteins with new termini in the β-sheet”, Prot. Sci., 2010, 19, 1490-1499.
Enter the name for this tabbed section: 36 Abstract
Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca2+ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca2+ biosensors.
Enter the name for this tabbed section: 35 Citation
35. R.E. Campbell and M.W. Davidson*, “Fluorescent reporter proteins”, Molecular Imaging with Reporter Genes. Eds. Sanjiv Sanjiv S. Gambhir and Shahriar S. Yaghoubi. Cambridge University Press, New York, NY, July 2010: 3 - 40.
Enter the name for this tabbed section: 35 Abstract
For more than a decade the growing class of fluorescent proteins (FPs), defined as homologues of Aequorea victoria green FP (avGFP) that are capable of forming an intrinsic chromophore, has almost single-handedly launched and fueled a new era in cell biology. These powerful research tools provide investigators with a means of fusing a genetically encoded optical probe to any one of a practically unlimited variety of protein targets in order to examine living systems using fluorescence microscopy and related methodology (see Figure 1; for recent reviews, see references [1-4]). The diverse array of practical applications for FPs ranges from targeted markers for organelles and other subcellular structures, to protein fusions designed to monitor mobility and dynamics, to reporters of transcriptional regulation (Figure 2). FPs have also opened the door to creating highly specific biosensors for live-cell imaging of numerous intracellular phenomena, including pH and ion concentration fluctuations, protein kinase activity, apoptosis, voltage, cyclic nucleotide signaling, and tracing neuronal pathways [5-9]. In addition, by applying selected promoters and targeting signals, FP biosensors can be introduced into an intact organism and directed to specific tissues, cell types, as well as subcellular compartments, to enable monitoring a variety of physiological processes using fluorescence resonance energy transfer (FRET) techniques.
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Enter the name for this tabbed section: 34 Citation
34. A. Ibraheem and R.E. Campbell*, “Designs and applications of fluorescent protein-based biosensors”, Current Opinion in Chemical Biology, 2010, 14, 30-36.
Enter the name for this tabbed section: 34 Abstract
Genetically encoded biosensors allow the noninvasive imaging of specific biochemical or biorecognition processes with the preservation of subcellular spatial and temporal information. Aequorea green fluorescent protein (FP) and its engineered variants are a critical component of genetically encoded biosensors, as they serve to provide a ‘read-out’ of the biorecognition event under investigation. The family of FP-based biosensors includes a diverse array of designs that utilize various photophysical characteristics of the FPs. In this review, we will discuss these designs and their read-outs through reviewing some of the recent works in this area.
Enter the name for this tabbed section: 33 Citation
33. R.E. Campbell* and C.J. Chang*, "Molecular Imaging: Editorial Overview", Current Opinion in Chemical Biology, 2010, 14, 1-2.
Enter the name for this tabbed section: 33 Abstract
Molecular Imaging, the creation and application of new approaches to study molecular-level events in biological systems, is a rapidly growing field that has attracted a broad base of researchers with diverse backgrounds spanning chemistry, physics, biology, medicine, and engineering. Perhaps more so than any other area of current scientific inquiry, Molecular Imaging offers a modern paradigm of how coordinated, collaborative efforts have advanced our understanding of medicine and the life sciences through fundamental inventions in the physical sciences and engineering. A major key to the success of these efforts is that the goals of Molecular Imaging are so readily appreciated by researchers from a disparate landscape of traditional disciplines. Chemists are motivated to directly visualize the location and action of specific molecules within complex living systems. Physicists and engineers embrace the theoretical and practical challenges of pushing imaging modalities to new extremes in order to obtain molecular information in both the spatial and temporal regimes with ever-higher resolution. Biologists and physicians, as the primary end users of Molecular Imaging technologies, are the major driving force for the continued advancement of tools for probing the chemistry of life at its most fundamental level.
This special issue of Current Opinion in Chemical Biology dedicated to Molecular Imaging seeks not only to present a cross-sectional overview of recent technological advances in the field, but to highlight specific examples of how novel tools for visualizing real-time events in complex living systems has and continues to enable researchers to gain critical new insights into important and long-standing biological problems. This synergy between technology and application continues to push the boundaries of how we understand the natural world around us and further drives the need for innovation in the development of new Molecular Imaging tools.
This special issue of Current Opinion in Chemical Biology dedicated to Molecular Imaging seeks not only to present a cross-sectional overview of recent technological advances in the field, but to highlight specific examples of how novel tools for visualizing real-time events in complex living systems has and continues to enable researchers to gain critical new insights into important and long-standing biological problems. This synergy between technology and application continues to push the boundaries of how we understand the natural world around us and further drives the need for innovation in the development of new Molecular Imaging tools.
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Served as co-editor (equal contributions) for this Special issue of the journal which had 15 invited reviews.
Served as co-editor (equal contributions) for this Special issue of the journal which had 15 invited reviews.
Lifetime numbering. Names of trainees in bold. Corresponding author(s) denoted with ‘*'.