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Enter the name for this tabbed section: 32 Citation
32. M.W. Davidson and R.E. Campbell*, “Engineered fluorescent proteins: innovations and applications”, Nature Methods, 2009, 6, 713-717.
Enter the name for this tabbed section: 32 Abstract
Despite expansion of the fluorescent protein and optical highlighter palette into the orange to far-red range of the visible spectrum, achieving performance equivalent to that of EGFP has continued to elude protein engineers.
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Invited Commentary for 5th Anniversary issue.
Enter the name for this tabbed section: 31 Citation
31. W.B. Frommer*, M.W. Davidson, R.E. Campbell* “Genetically encoded biosensors based on engineered fluorescent proteins”, Chemical Society Reviews, 2009, 38, 2833-2841.
Enter the name for this tabbed section: 31 Abstract
Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.
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Supplement: a referenced list of most of the FP-based biosensors reported to time of publication.
Enter the name for this tabbed section: 30 Citation
30. D.E. Johnson, H-w. Ai, P. Wong, J.D. Young, R.E. Campbell, and J.R. Casey* “A red fluorescent protein pH biosensor for detection of concentrative nucleoside transport”, Journal of Biological Chemistry, 2009, 284, 20499-20511.
Enter the name for this tabbed section: 30 Abstract
Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+ (1.09 ± 0.11 or 1.18 ± 0.32 mM min−1, respectively). This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H+-coupled transport proteins.
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Enter the name for this tabbed section: 29 Citation
29. R.E. Campbell*, “Fluorescent Protein-Based Biosensors: Modulation of Energy Transfer as a Design Principle”, Analytical Chemistry, 2009, 81(15): 5972–5979.
Enter the name for this tabbed section: 29 Abstract
Genetically-encoded biosensors based on FRET between fluorescent proteins of different hues enable quantitative measurement of intracellular enzyme activities and small molecule concentrations.
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Invited Feature article with cover art and accompanying podcast (Aug 01, 2009).
Enter the name for this tabbed section: 28 Citation
28. Z. Cheng and R.E. Campbell*, “An engineered tryptophan zipper-type peptide as a molecular recognition scaffold”, Journal of Peptide Science, 2009, 15, 523-532.
Enter the name for this tabbed section: 28 Abstract
In an effort to develop a structured peptide scaffold that lacks a disulfide bond and is thus suitable for molecular recognition applications in the reducing environment of the cytosol, we investigated engineered versions of the trpzip class of β-hairpin peptides. We have previously shown that even most highly folded members of the trpzip class (i.e. the 16mer peptide HP5W4) are substantially destabilized by the introduction of mutations in the turn region and therefore not an ideal peptide scaffold. To address this issue, we used a FRET-based live cell screening system to identify extended trpzip-type peptides with additional stabilizing interactions. One of the most promising of these extended trpzip-type variants is the 24mer xxtz1-peptide with the sequence KAWTHDWTWNPATGKWTWLWRKNK. A phage display library of this peptide with randomization of six residues with side chains directed towards one face of the hairpin was constructed and panned against immobilized streptavidin. We have also explored the use of xxtz1-peptide for the presentation of an unstructured peptide ‘loop’ inserted into the turn region. Although NMR analysis provided no direct evidence for structure in the xxtz1-peptide with the loop insertion, we did attempt to use this construct as a scaffold for phage display of randomized peptide libraries. Panning of the resulting libraries against streptavidin resulted in the identification of peptide sequences with submicromolar affinities. Interestingly, substitution of key residues in the hairpin-derived portion of the peptide resulted in a 400-fold decrease in Kd, suggesting that the hairpin-derived portion plays an important role in preorganization of the loop region for molecular recognition.
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Enter the name for this tabbed section: 27 Citation
27. H.J. Carlson, R.E. Campbell*, “Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging”, Current Opinion in Biotechnology, 2009, 20: 19-27.
Enter the name for this tabbed section: 27 Abstract
The phenomenon of Förster (or fluorescence) resonance energy transfer (FRET) between two fluorescent proteins of different hues provides a robust foundation for the design and construction of biosensors for the detection of intracellular events. Accordingly, FRET-based biosensors for a variety of biologically relevant ions, molecules, and specific enzymatic activities, have now been developed and used to investigate numerous questions in cell biology. An emerging trend in the use of FRET-based biosensors is to apply them in combination with a second biosensor in order to achieve simultaneous imaging of multiple biochemical parameters in a single living cell. Here we discuss the particular technological challenges facing the use of FRET-based biosensors in multiparameter live cell fluorescence imaging and highlight recent efforts to overcome these challenges. In addition, we survey recent applications and provide an outlook on the future opportunities in this area.
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Enter the name for this tabbed section: 26 Citation
26. R.E. Campbell*, “Fluorescent proteins”, Scholarpedia, 2008, 3(7): 5410.
Enter the name for this tabbed section: 26 Abstract
Fluorescent proteins are members of a structurally homologous class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of 3 amino acids within their own polypeptide sequence. It is common research practice for biologists to introduce a gene (or a gene chimera) encoding an engineered fluorescent protein into living cells and subsequently visualize the location and dynamics of the gene product using fluorescence microscopy.
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Enter the name for this tabbed section: 25 Citation
25. H-w. Ai, K.L. Hazelwood, M.W. Davidson, and R.E. Campbell*, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors”, Nature Methods, 2008, 5, 401-403.
Enter the name for this tabbed section: 25 Abstract
Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET–based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.
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Supplementary movies

Cover story of October 2008 issue of Biophotonics.
Enter the name for this tabbed section: 24 Citation
24. H-w. Ai, S.G. Olenych, P. Wong, M.W. Davidson, and R.E. Campbell*, “Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging”, BMC Biology, 2008, 6: 13.
Enter the name for this tabbed section: 24 Abstract
In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.

Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins.

Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.
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Designated Highly Accessed and Featured article.
Enter the name for this tabbed section: 23 Citation
23. H-w. Ai, and R. E. Campbell*, “Teal fluorescent proteins: Characterization of a reversibly photoconvertible variant”, Proceedings of SPIE, 2008, 6868, 68680D.
Enter the name for this tabbed section: 23 Abstract
Fluorescent proteins (FPs) emerged in the mid 1990s as a powerful tool for life science research. Cyan FPs (CFPs), widely used in multicolor imaging or as a fluorescence resonance energy transfer (FRET) donor to yellow FPs (YFPs), were considerably less optimal than other FPs because of some relatively poor photophysical properties. We recently initiated an effort to create improved or alternate versions of CFPs. To address the limitations of CFPs, an alternative known as monomeric teal FP1 (mTFP1) was engineered from the naturally tetrameric Clavularia CFP, by screening either rationally designed or random libraries of variants. mTFP1 has proven to be a particularly useful new member of the FP ‘toolbox’ by facilitating multicolor live cell imaging. During the directed evolution process of mTFP1, it was noticed that some earlier variants underwent fast and reversible photoisomerization. Some of the initial characterization of one particular mutant, designated as mTFP0.7, is described in this manuscript.
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Enter the name for this tabbed section: 22 Citation
22. Q.K. Timerghazin, H.J. Carlson, C. Liang, R.E. Campbell,* and A. Brown*, “Computational prediction of absorbance maxima for a structurally diverse series of engineered green fluorescent protein chromophores”, J. Phys. Chem B, 2008, 112: 2533-2541.
Enter the name for this tabbed section: 22 Abstract
By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea Victoria green fluorescent protein (GFP) is single-handedly responsible for the ever- growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the experimental values. Interestingly, most of the computational methods used in this work fail in the case of nonaromatic Y66S and Y66L protein chromophores, which may be related to a significant contribution of double excitations to their excited-state wavefunctions. These results provide an important benchmark of the reliability of the computational methods as applied to GFP chromophore analogues and lays a foundation for the computational design of GFP variants with improved properties for use in biological imaging.
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Enter the name for this tabbed section: 21 Citation
21. Y. Li, A.M. Sierra, H.-w. Ai, and R.E. Campbell*, “Identification of sites within a monomeric red fluorescent protein that tolerate peptide insertion and testing of corresponding circular permutations”, Photochemistry and Photobiology, 2008, 84: 111–119.
Enter the name for this tabbed section: 21 Abstract
In recent years the class of known fluorescent proteins (FPs) has dramatically expanded as an ever-increasing numbers of variants and homologs of the green fluorescent protein (GFP) from Aequorea jellyfish have been either engineered in the lab or discovered in other marine organisms. The red fluorescent protein (RFP) from Discosoma coral (also known as dsFP583 and DsRed) has proven to be a particularly fruitful progenitor of variants with biochemical and spectroscopic properties conducive to applications in live cell imaging. We have investigated the tolerance of an engineered monomeric descendent of Discosoma RFP, known as mCherry, towards peptide insertion and circular permutation. Starting from a random library of insertion variants, we identified six genetically distinct sites localized in three different loops where a sequence of five residues could be inserted without abolishing the ability of the protein to form its intrinsic red fluorescent chromophore. For each of these insertion variants, a corresponding circular permutation variant was created in which the original N- and C-termini were connected by a six-residue linker and new termini were introduced at the site of the insertion. All six circular permutation variants had significantly diminished brightness relative to the analogous insertion variants. The most promising circular permutation variant has termini at the position corresponding to residue 184 of mCherry and retains 37% of the intrinsic fluorescent brightness of mCherry. These circularly permuted variants may serve as the foundation for construction of genetically encoded Ca2+ sensors analogous to the previously reported camgaroo, pericam and G-CaMP sensors based on variants of Aequorea GFP.
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Enter the name for this tabbed section: 20 Citation
20. H-w. Ai and R.E. Campbell*, “More than just pretty colors: the growing impact of fluorescent proteins in the life sciences”, Biotechnology Focus, 2007, issue 11: 16-18.
Enter the name for this tabbed section: 20 Abstract
How is it that a jellyfish sparked a revolution in biotechnology? The year was 1962 when a Princeton University researcher by the name of Osamu Shimomura reported on the purification and characterization of the protein responsible for the bioluminescence of Aequorea jellyfish. Shimomura had painstakingly harvested many thousands of jellyfish, cut off its bioluminescent organs with scissors and squeezed the proteins from the tissue wrapped in a handkerchief: a procedure that produced a solution known aptly as ‘squeezate’. From this squeezate Shimomura isolated the bioluminescent protein aequorin; a tremendous accomplishment in its own right. In a footnote within the manuscript describing this work, he mentions the presence of another curious protein in squeezate that was not bioluminescent but rather fluorescent.
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Enter the name for this tabbed section: 19 Citation
19. Z. Cheng, M. Miskolzie, and R.E. Campbell*, “In vivo screening identifies a highly folded beta-hairpin peptide with a structured extension”, ChemBioChem, 2007, 8: 880-883.
Enter the name for this tabbed section: 19 Abstract
Like finding a hairpin in the haystack. We have used an in vivo FRET-based screening method to identify highly folded β-hairpin peptides in large libraries. An NMR structure reveals that a cross-strand cation–π interaction helps stabilize the most highly folded β-hairpin.
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Supporting Information

Featured with cover art.
Enter the name for this tabbed section: 18 Citation
18. H.-w. Ai, N.C. Shaner, Z. Cheng, R.Y. Tsien, and R.E. Campbell*, “Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins”, Biochemistry, 2007, 46: 5904 - 5910.
Enter the name for this tabbed section: 18 Abstract
The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein “toolkit”.
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News story featured on the cover of June 2007 issue of Biophotonics.
9th Most-accessed article in Biochemistry for 2007.
Enter the name for this tabbed section: 17 Citation
17. J.N. Henderson, H.-w. Ai, R.E. Campbell, and S.J. Remington*, “Structural basis for reversible photobleaching of a green fluorescent protein homologue”, Proc. Natl. Acad. Sci. U.S.A., 2007, 14: 6672-6677.
Enter the name for this tabbed section: 17 Abstract
Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cis–trans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.
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Supporting Information

News story in June 2007 issue of Biophotonics and April 2007 Science Daily online.
Enter the name for this tabbed section: 16 Citation
16. Z. Cheng and R.E. Campbell*, “Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress towards a generic hairpin scaffold”, Proceedings of SPIE, 2007, 6449, 64490S.
Enter the name for this tabbed section: 16 Abstract
Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.
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Enter the name for this tabbed section: 15 Citation
15. H-w. Ai, J.N. Henderson, S.J. Remington, and R.E. Campbell*, “Directed evolution of a monomeric, bright, and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging”, Biochem. J., 2006, 400: 531-540.
Enter the name for this tabbed section: 15 Abstract
The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 Å crystal structure (1 Å=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
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Enter the name for this tabbed section: 14 Citation
14. Z. Cheng and R.E. Campbell*, “Assessing the Structural Stability of Designed β-Hairpin Peptides in the Cytoplasm of Live Cells”, ChemBioChem, 2006, 7: 1147-1150.
Enter the name for this tabbed section: 14 Abstract
A genetically encoded β-hairpin peptide molecular beacon. By exploiting FRET between genetically fused fluorescent protein variants, we have determined the relative stability of a series of β-hairpins both in vivo and in vitro.
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Enter the name for this tabbed section: 13 Citation
13. R.E. Campbell*, “Realization of β-lactamase as a versatile fluorogenic reporter”, Trends Biotech., 2004, 22: 208-211.
Enter the name for this tabbed section: 13 Abstract
β-Lactamase has emerged as the heir apparent to β-galactosidase as a catalytic reporter for imaging biological events in live mammalian cells. In recent years, several publications have demonstrated the advantages of β-lactamase as a reporter in applications ranging from monitoring of gene transcription to detection of protein–protein interactions. Now, Rao et al. have demonstrated that β-lactamase can also serve as a sensitive fluorogenic reporter for imaging Tetrahymena ribozyme activity in live cells. This assay should pave the way for the screening of large libraries of ribozyme mutants by flow cytometry and, therefore, the isolation of variants with improved splicing activity in the cytoplasm of mammalian cells.
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Lifetime numbering. Names of trainees in bold. Corresponding author denoted with ‘*'.