Acta Crystallographica Section D

Biological Crystallography

Volume 69, Part 12 (December 2013)

research papers

Acta Cryst. (2013). D69, 2513-2523    [ doi:10.1107/S0907444913024608 ]

Split green fluorescent protein as a modular binding partner for protein crystallization

H. B. Nguyen, L.-W. Hung, T. O. Yeates, T. C. Terwilliger and G. S. Waldo

Abstract: A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP [beta]-­strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

PDB references: 4kf4 and 4kf5

Keywords: protein crystallization; synthetic symmetrization; protein tagging; split GFP; split protein; green fluorescent protein; protein expression; protein-fragment complementation; crystallization reagents.

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[ doi:10.1107/S0907444913024608/dz5297sup1.pdf ]
Supplementary figures and table.


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