研究领域
Nature has provided many proteins with catalytic, structural, or physical properties that render them useful as tools in chemical, biochemical, or biotechnological applications. However, the evolutionary forces that have generated this myriad of proteins are very different from the quite unnatural demands humans place on their tools. It is often necessary to "improve" natural proteins to make them better tools. For example, to be useful for industrial applications an enzyme may have to be engineered to be stable and active at high temperature or in the presence of solvents that a protein would not encounter in Nature. As difficult as it may be to generate ideal 'protein-based' tools, the effort is justified because proteins can have such remarkable structural (e.g. spider silk), catalytic (enzymes), or physical (e.g. green fluorescent protein) properties. It is for this reason that Protein Engineering, i.e. the directed evolution of existing proteins or the creation of de novo proteins with no structural or functional homologue in Nature, is a critical direction of research.
My specific focus is on the use of Protein Engineering for the development of genetically encoded fluorescent labels and reporters for imaging and manipulation of biochemistry in living cells. Fluorescent proteins (FPs), such as the Aequorea jellyfish green fluorescent protein (GFP) (see Figure 1), are nearly ideal fluorescent labels because they can be expressed in a variety of different organisms and fused to many different proteins of interest with little or no effect on either proteins function. Reporters based on fluorescence resonance energy transfer (FRET) between two engineered variants of GFP, a cyan FP (CFP) and a yellow FP (YFP), have found great utility in cell biology (Figure 2A). An ideal complement to the numerous reporters of this type would be a spectrally distinct red-shifted FRET pair that would allow simultaneous imaging of two reporters to determine causal relationships between biochemical processes. Efforts to develop such a FRET pair, using both red-shifted FPs and in situ labeling strategies, will be a major focus of my research. An alternative reporter design is to engineer FP variants in which binding of a small molecule directly modulates the fluorescence spectrum (Figure 2B). This type of sensor has been reported but current designs lack generality. To overcome this limitation I plan to employ Protein Engineering to create de novo sensors that could be tailored to detect any small molecule of interest.
While the chemical composition of proteins is rather simple (a linear polypeptide composed of the 20 common amino acids) their 3-dimensional structures are fantastically complex. However, moderate modifications of complex protein structures such as point mutations, insertions and deletions, or fusion to another protein, are simple procedures that can be achieved in just a few hours of work using the techniques of molecular biology. The challenge of Protein Engineering is to use these simple molecular biology techniques to produce a new protein that is either an improvement over the original or has acquired some completely new function. Generally speaking, we have not yet reached the point where we can predict what effect a given point mutation will have on a given protein's structure and function. For this reason, the most effective approach to Protein Engineering is to rely on high throughput screening of libraries of mutated proteins with selection of those rare variants that are improved over the parent protein. Methods of screening can range from very low throughput (manual assay of individual clones), to medium throughput (96-well plate format or screening of bacterial colonies), to very high throughput (phage display and fluorescence activated cell sorting (FACS)). Research in my laboratory will be highly multidisciplinary with projects that involve organic synthesis, molecular biology, protein crystallography, molecular modeling, and live cell fluorescence microscopy. I anticipate that in the course of their graduate research a typical student will obtain a thorough training in at least two of the above disciplines, will be very familiar with the others, and will have a built a strong foundation on which to start a successful and rewarding career in chemistry, biochemistry, or biotechnology.
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Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations T. Albrecht (equal contribution), Y. Zhao (equal contribution), T.H. Nguyen, R.E. Campbell, and J.D. Johnson*, “Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations”, Cell Calcium, 2015. Accepted January 19, 2015.
Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange Y. Ding, J. Li, J.R. Enterina, Y. Shen, I. Zhang, P.H. Tewson, G.C.H. Mo, J. Zhang, A.M. Quinn, T.E. Hughes, D. Maysinger, S.C. Alford, Y. Zhang, and R.E. Campbell*, “Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange”, Nat. Methods, 2015. Published online January 26, 2015.
A Photochromic and Thermochromic Fluorescent Protein Y. Shen, M.D. Wiens, and R.E. Campbell*, “A Photochromic and Thermochromic Fluorescent Protein”. RSC Adv., 2014, 4, 56762-56765. [Open Access PDF]
pHuji, a pH sensitive red fluorescent protein for imaging of exo- and endocytosis Y. Shen (equal contribution), M. Rosendale (equal contribution), R.E. Campbell (*correspondance related to new FP variants), and D. Perrais*, “pHuji, a pH sensitive red fluorescent protein for imaging of exo- and endocytosis”, J. Cell Biol., 2014, 207 (3): 419-432.
A long Stokes shift red fluorescent protein Ca2+ indicator for 2-photon and ratiometric imaging J. Wu, A.S. Abdelfattah, L.S. Miraucourt, E. Kutsarova, A. Ruangkittisakul, H. Zhou, K. Ballanyi, G. Wicks, M. Drobizhev, A. Rebane, E.S. Ruthazer, and R.E. Campbell*, “A long Stokes shift red fluorescent protein Ca2+ indicator for 2-photon and ratiometric imaging”, Nat. Commun., 2014, 5, 5262. [Supplementary Material; Funding from NSERC Discovery, CIHR MOP 123514, and a Vanier Canada Graduate and Alberta Innovates Health Solutions (AIHS) Scholarships to A.S.A]
Excited State Structural Events of a Dual-Emission Fluorescent Protein Biosensor for Ca2+ Imaging Studied by Femtosecond Stimulated Raman Spectroscopy Y. Wang, L. Tang, W. Liu, Y. Zhao, B.G. Oscar, R.E. Campbell, and C. Fang*, “Excited State Structural Events of a Dual-Emission Fluorescent Protein Biosensor for Ca2+ Imaging Studied by Femtosecond Stimulated Raman Spectroscopy”, J. Phys. Chem. B, 2014, Article ASAP, Published online September 16, 2014.
Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum J. Wu, D.L. Prole, Y. Shen, Z. Lin, A. Gnanasekaran, Y. Liu, L. Chen, H. Zhou, S.R.W. Chen, Y.M. Usachev, C.W. Taylor, and R.E. Campbell*, “Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum”, Biochem. J., 2014, 464, 13–22. [Supplementary Material; Funding from NSERC Discovery, CIHR MOP 123514, and a graduate scholarship from Alberta Innovates to Y.S.]
Bright and fast multi-colored voltage reporters via electrochromic FRET P. Zou (equal contribution), Y. Zhao (equal contribution), A.D. Douglass, D.R. Hochbaum, D. Brinks, C.A. Werley, D.J. Harrison, R.E. Campbell (*correspondence regarding the library screen), A.E. Cohen*, “Bright and fast multicolored voltage reporters via electrochromic FRET”, Nat. Commun., 2014, 5, 4625. [Supplementary Material; Funding from NSERC Discovery, CIHR MOP 123514, and graduate scholarships from the University of Alberta and Alberta Innovates to Y.Z.]
Excited state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging B.G. Oscar, W. Liu, Y. Zhao, L. Tang, Y Wang, R.E. Campbell, and C. Fang*, “Excited state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging”, Proc. Natl. Acad. Sci. U.S.A., 2014, 111, 10191–10196. [Supplementary Material; Funding from NSERC Discovery, CIHR MOP 123514, and graduate scholarships from the University of Alberta and Alberta Innovates to Y.Z.]
All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins D.R. Hochbaum (equal contribution), Y. Zhao (equal contribution), S.L. Farhi, N. Klapoetke, C.A. Werley, V. Kapoor, P. Zou, J.M. Kralj, D. Maclaurin, N. Smedemark-Margulies, J. Saulnier, G.L. Boulting, Y. Cho, M. Melkonian, G.K-S. Wong, D.J. Harrison, V.N. Murthy, B. Sabatini, E.S. Boyden (equal contribution), R.E. Campbell (equal contribution; *correspondance related to directed evolution), and A.E. Cohen*, “All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins”, Nat. Methods, 2014, 11, 825–833. [Supplementary Material;Sample requests; Funding from NSERC Discovery, CIHR MOP 123514, and graduate scholarships from the University of Alberta and Alberta Innovates to Y.Z.; Highlighted by Science Media Centre of Canada]
Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response Y. Zhao, A.S. Abdelfattah, Y. Zhao, A. Ruangkittisakul, K. Ballanyi, R.E. Campbell*, D.J. Harrison*, “Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response”, Integr. Biol. (Camb), 2014, 6(7), 714-725. [Open Access PDF; Supplementary material, Movie 1, Movie 2; Funding from NSERC Discovery, CIHR MOP 123514, and graduate scholarships from the University of Alberta (Y.Z) and Alberta Innovates to (Y.Z. and A.S.A)]
Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications 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, 9, 910-928. [Funding from University of Alberta, CFI, NSERC Discovery grant, and Alberta Ingenuity (Scholarship to Y.S. and a New Faculty Award to R.E.C.)]
Optimization of a Genetically Encoded Biosensor for Cyclin B1-Cyclin Dependent Kinase 1 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. [Open Access PDF; Supplementary material; Funded by NSERC]
FRET with Fluorescent Proteins H. Hoi, Y. Ding, and R.E. Campbell*, “FRET with Fluorescent Proteins”, in FRET – Förster Resonance Energy Transfer: From Theory to Applications. Eds. Igor Medintz and Niko Hildebrandt. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, November 2013, pages 431-473. [Google book preview; Funded by NSERC Discovery and CIHR NHG 99085]
An engineered monomeric Zoanthus sp. yellow fluorescent protein 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. [Highlighted in the same issue; Supplementary Material; Funded by NSERC Discovery and Alberta Innovates Technology Futures (AITF) Scholarship to W.Z.]
Mutational analysis of a red fluorescent protein-based calcium ion indicator H.J. Carlson and R.E. Campbell*, “Mutational analysis of a red fluorescent protein-based calcium ion indicator”, Sensors, 2013, 13(9), 11507-11521. [Open Access PDF; Supplementary Material; Funded by NSERC Discovery, NSERC PGSM, and Alberta Ingenuity Scholarship]
Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry 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(12): 763-772. [Supplementary Material; Funded by NSERC Discovery, NSERC PGSM, and Alberta Ingenuity Scholarship]
Palmitoylation is the Switch that Assigns Calnexin to Quality Control or ER Calcium Signaling 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. [Supplementary Material; Funded by CIHR NHG 99085]
Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications 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(6), 963-972. [Supplementary Material; Funded by CIHR NHG 99085, CIHR MOP 123514, NSERC Discovery, and Alberta Ingenuity Nanotechnology Scholarship to Y.Z.; Highlighted at OpenOptogenetics]
Highlightable Ca2+ indicators for live cell imaging H. Hoi, T. Matsuda, T. Nagai, and R.E. Campbell*, “Highlightable Ca2+ indicators for live cell imaging”, J. Am. Chem. Soc., 2013, 135(1), 46-49. [Supplementary Material; Funded by NSERC Discovery; Highlighted at OpenOptogenetics]
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