Jyoti Kumari, J. and Surbhi Dhingra, S. and Janesh Kumar, J. Fluorescence-Based Screening of Membrane Proteins for Structural Studies. In: Shukla A. (eds) Chemical and Synthetic Approaches in Membrane Biology. In: Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks book series (SPH) . Springer, pp. 141-155.

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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.

Item Type:	Book Section
Subjects:	Structural Biology
Depositing User:	Mr. Rameshwar Nema
Date Deposited:	05 Dec 2017 10:46
Last Modified:	05 Dec 2017 10:46
URI:	http://nccs.sciencecentral.in/id/eprint/486

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