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Fluorescent Proteins: Crystallization, Structural Determination, and Nonnatural Amino Acid Incorporation

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Fluorescent Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2564))

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Abstract

Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties. Here we describe the process by which fluorescent protein structures are determined, starting from recombinant protein production to structure determination by molecular replacement. We also describe how to incorporate and determine the structures of proteins containing non-natural amino acids. Recent advances in protein engineering have led to reprogramming of the genetic code to allow incorporation of new chemistry at designed residue positions, with fluorescent proteins being at the forefront of structural studies in this area. The impact of such new chemistry on protein structure is still limited; the accumulation of more protein structures will undoubtedly improve our understanding and ability to engineer proteins with new chemical functionality.

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Acknowledgments

We would like to thank the staff at the Diamond Light Source (Harwell, UK) for the supply of facilities and beam time, especially beamlines I02, I03, and I04 staff, under beamtime code mx18812. This work was supported by BBSRC (BB/H003746/1 and BB/M000249/1) and EPSRC (EP/ J015318/1) grants (to DDJ). RDA was supported by KESS 2-Tenovus studentship and HSA by the Higher Committee for Education Development in Iraq. We would like to thank the Protein Technology Hub, School of Biosciences, Cardiff University, for use of facilities to generate protein and analyze protein essential to structural studies. We would also like to thank Ben Bax and Magdalena Lipka-Lloyd for help with protein crystallization. Finally, we are eternally grateful to Pierre Rizkallah for helping with all aspects of protein crystallography over the years and teaching many members of the DDJ group how to determine protein structures—we salute you, sir.

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Authors and Affiliations

  1. School of Biosciences, Molecular Biosciences Division, Cardiff University, Cardiff, UK

    Rochelle D. Ahmed & D. Dafydd Jones

  2. Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq

    Husam Sabah Auhim

  3. Department of Biosciences, University of Exeter, Exeter, UK

    Harley L. Worthy

Authors
  1. Rochelle D. Ahmed
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  2. Husam Sabah Auhim
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  3. Harley L. Worthy
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  4. D. Dafydd Jones
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Corresponding author

Correspondence to D. Dafydd Jones .

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Editors and Affiliations

  1. Institute of Molecular Plant Biology, ETH Zürich, Zürich, Switzerland

    Mayank Sharma

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Ahmed, R.D., Auhim, H.S., Worthy, H.L., Jones, D.D. (2023). Fluorescent Proteins: Crystallization, Structural Determination, and Nonnatural Amino Acid Incorporation. In: Sharma, M. (eds) Fluorescent Proteins. Methods in Molecular Biology, vol 2564. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2667-2_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2667-2_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2666-5

  • Online ISBN: 978-1-0716-2667-2

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