title: Split green fluorescent protein as a modular binding partner for protein crystallization.
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 β-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.
authors: Bioscience Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545, USA.
authors: Hau B
authors: HB
authors: Nguyen
authors: Li-Wei
authors: LW
authors: Hung
authors: Todd O
authors: TO
authors: Yeates
authors: Thomas C
authors: TC
authors: Terwilliger
authors: Geoffrey S
authors: GS
authors: Waldo
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray / Q000379:methods; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
keywords: crystallization reagents; green fluorescent protein; protein crystallization; protein expression; protein tagging; protein-fragment complementation; split GFP; split protein; synthetic symmetrization