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

Nguyen HB, Hung LW, Yeates TO, Terwilliger TC, Waldo GS - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioscience Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545, USA.

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.

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A 2mFo − DFc σA-weighted electron-density map (Winn et al., 2011 ▶) was calculated using a model that was constructed before any connections between sfCherry and the GFP(10–11) hairpin had been built. The connections between the green arrows (not included in the phasing model) are between residues 168 and 172 and residues 205 and 211. This unbiased map (contoured at 0.5σ in gray and at 1σ in blue) shows clear connections between sfCherry and the GFP(10–11) hairpin that was inserted into sfCherry at an exposed loop.
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fig5: A 2mFo − DFc σA-weighted electron-density map (Winn et al., 2011 ▶) was calculated using a model that was constructed before any connections between sfCherry and the GFP(10–11) hairpin had been built. The connections between the green arrows (not included in the phasing model) are between residues 168 and 172 and residues 205 and 211. This unbiased map (contoured at 0.5σ in gray and at 1σ in blue) shows clear connections between sfCherry and the GFP(10–11) hairpin that was inserted into sfCherry at an exposed loop.

Mentions: The structure of sfCherry-GFP(10–11) in complex with GFP(1–9) was determined at 2.6 Å resolution, with final R and Rfree values of 0.205 and 0.247, respectively (Table 1 ▶). No major elements of disorder, conformational heterogeneity or anisotropy were observed. The structure of the complex (Fig. 4 ▶b) shows sfCherry to be clearly linked to the GFP(10–11) hairpin and that the GFP(10–11) hairpin complements GFP(1–9) to form an intact GFP molecule. The crystal asymmetric unit contains two copies of the complex. With two complexes in the asymmetric unit, and two linking chain segments between the two protein components in each case, there are four linking polypeptide segments. All of these segments are well ordered and clearly visible in the final electron-density map (Fig. 5 ▶). Furthermore, the relative orientation of the GFP and sfCherry components in the complex is very similar in the two instances visualized in the asymmetric unit. When the GFP components of the two independent complexes are spatially overlapped, the sfCherry components differ in the two cases by a rotation of only 9° (Fig. 6 ▶).


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

Nguyen HB, Hung LW, Yeates TO, Terwilliger TC, Waldo GS - Acta Crystallogr. D Biol. Crystallogr. (2013)

A 2mFo − DFc σA-weighted electron-density map (Winn et al., 2011 ▶) was calculated using a model that was constructed before any connections between sfCherry and the GFP(10–11) hairpin had been built. The connections between the green arrows (not included in the phasing model) are between residues 168 and 172 and residues 205 and 211. This unbiased map (contoured at 0.5σ in gray and at 1σ in blue) shows clear connections between sfCherry and the GFP(10–11) hairpin that was inserted into sfCherry at an exposed loop.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852656&req=5

fig5: A 2mFo − DFc σA-weighted electron-density map (Winn et al., 2011 ▶) was calculated using a model that was constructed before any connections between sfCherry and the GFP(10–11) hairpin had been built. The connections between the green arrows (not included in the phasing model) are between residues 168 and 172 and residues 205 and 211. This unbiased map (contoured at 0.5σ in gray and at 1σ in blue) shows clear connections between sfCherry and the GFP(10–11) hairpin that was inserted into sfCherry at an exposed loop.
Mentions: The structure of sfCherry-GFP(10–11) in complex with GFP(1–9) was determined at 2.6 Å resolution, with final R and Rfree values of 0.205 and 0.247, respectively (Table 1 ▶). No major elements of disorder, conformational heterogeneity or anisotropy were observed. The structure of the complex (Fig. 4 ▶b) shows sfCherry to be clearly linked to the GFP(10–11) hairpin and that the GFP(10–11) hairpin complements GFP(1–9) to form an intact GFP molecule. The crystal asymmetric unit contains two copies of the complex. With two complexes in the asymmetric unit, and two linking chain segments between the two protein components in each case, there are four linking polypeptide segments. All of these segments are well ordered and clearly visible in the final electron-density map (Fig. 5 ▶). Furthermore, the relative orientation of the GFP and sfCherry components in the complex is very similar in the two instances visualized in the asymmetric unit. When the GFP components of the two independent complexes are spatially overlapped, the sfCherry components differ in the two cases by a rotation of only 9° (Fig. 6 ▶).

Bottom Line: A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioscience Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545, USA.

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.

Show MeSH
Related in: MedlinePlus