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Fluorescence-Based Screening of Membrane Proteins for Structural Studies

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Chemical and Synthetic Approaches in Membrane Biology

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Abstract

Structure determination of membrane proteins remains a technically challenging problem till date. Optimization of expression, purification, and stability of membrane proteins is still a bottleneck for their structure determination by either X-ray crystallography or single-particle cryo-electron microscopy. Their expression levels are low; hydrophobic transmembrane domains require detergents to extract them from cell membrane. Besides this, they are often flexible and unstable and must be stabilized before subjecting them to structure determination trials. Traditional methods of screening either require large amounts of material or are limited in their use due to the presence of detergents, which hinder with or cause high background during these evaluations.

Here, we describe two fluorescence-based methods that have enabled rapid, efficient, and economical screening of membrane proteins for construct optimization and evaluation of suitable stability conditions essential for successful structure determination. Both the techniques require only nanogram to microgram quantities of material and can be adapted to perform medium- to high-throughput screening.

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Acknowledgments

The authors acknowledge financial assistance from the University Grants Commission (UGC), New Delhi (J.K.), and the Department of Biotechnology, New Delhi. This work was supported by the Wellcome Trust/DBT India Alliance intermediate fellowship to Janesh Kumar.

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Authors and Affiliations

  1. National Centre for Cell Science, NCCS Complex, S. P. Pune University Campus, Ganeshkhind, Pune, Maharashtra, 411007, India

    Jyoti Kumari, Surbhi Dhingra & Janesh Kumar

Authors
  1. Jyoti Kumari
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  2. Surbhi Dhingra
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  3. Janesh Kumar
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Corresponding author

Correspondence to Janesh Kumar .

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Editors and Affiliations

  1. Indian Institute of Technology, Kanpur, India

    Arun K. Shukla

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Kumari, J., Dhingra, S., Kumar, J. (2016). Fluorescence-Based Screening of Membrane Proteins for Structural Studies. In: Shukla, A. (eds) Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/8623_2016_1

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  • DOI: https://doi.org/10.1007/8623_2016_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6835-0

  • Online ISBN: 978-1-4939-6836-7

  • eBook Packages: Springer Protocols

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