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

A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. 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:
 [1] ;  [2] ;  [3] ; ;  [1]
  1. Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545 (United States)
  2. Los Alamos National Laboratory, MS D454, Los Alamos, NM 87545 (United States)
  3. University of California, PO Box 951569, Los Angeles, CA 90095 (United States)
Publication Date:
OSTI Identifier:
22347805
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 69; Journal Issue: Pt 12; Other Information: PMCID: PMC3852656; PMID: 24311592; PUBLISHER-ID: dz5297; OAI: oai:pubmedcentral.nih.gov:3852656; Copyright (c) Nguyen et al. 2013; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTAL STRUCTURE; CRYSTALLIZATION; CRYSTALS; JOINTS; PROTEINS; RESOLUTION; SURFACES