Limits...
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.

Show MeSH

Related in: MedlinePlus

Principle of the work: insertion of GFP hairpin strands S10 and S11 into a permissive loop of a target protein, followed by reconstitution of the intact GFP by attachment of GFP(1–9) (i.e. the GFP molecule missing the hairpin).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3852656&req=5

fig1: Principle of the work: insertion of GFP hairpin strands S10 and S11 into a permissive loop of a target protein, followed by reconstitution of the intact GFP by attachment of GFP(1–9) (i.e. the GFP molecule missing the hairpin).

Mentions: The structure, stability and folding of GFP have been well studied (Örmo et al., 1996 ▶; Tsien, 1998 ▶; Crameri et al., 1996 ▶). Its relatively simple topology, combined with its utility as a fluorescent reporter when correctly folded (Waldo et al., 1999 ▶; Pédelacq et al., 2006 ▶), has made it an attractive system for reconstitution from separately expressed protein fragments (Cabantous, Terwilliger et al., 2005 ▶). Following such a strategy, by fusing terminal segments of GFP to a crystallization target the resulting construct might be recombined with the remaining complementary fragment of GFP to create a new complex for crystallization. In the context of crystallization strategies, a challenge presented by typical fusion methods is the flexibility introduced at the site of connection between the two protein components; free torsion angles are present where the polypeptide backbone makes its (single) crossing from one natural protein fold to the other. The value of having the polypeptide chain cross twice instead of once between two connected proteins has been demonstrated in experiments in which T4 lysozyme was inserted into a loop of GPCR membrane proteins, giving a construct that yielded well ordered crystals (Rosenbaum et al., 2007 ▶; Cherezov et al., 2007 ▶). The split GFP system [GFP(1–9) + GFP(10–11)] allows a similar advantage. If strands 10 and 11, which ostensibly form a natural hairpin, can be inserted as a long extension into a surface loop of a target protein, then reconstitution with complementary GFP(1–9) should give a tight noncovalent complex with two chain crossings between natural protein folds (Fig. 1 ▶). In practice, rational choices for the points of insertion of strands 10–11 into exposed loops might be based on homology models, where available, or on bioinformatic predictions of loops (Lambert et al., 2002 ▶; Dovidchenko et al., 2008 ▶; Jones, 1999 ▶). Here, we chose a target for crystallization for which the structure was known, in order to test the strategy of loop insertion and crystallization in a favorable case.


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)

Principle of the work: insertion of GFP hairpin strands S10 and S11 into a permissive loop of a target protein, followed by reconstitution of the intact GFP by attachment of GFP(1–9) (i.e. the GFP molecule missing the hairpin).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Principle of the work: insertion of GFP hairpin strands S10 and S11 into a permissive loop of a target protein, followed by reconstitution of the intact GFP by attachment of GFP(1–9) (i.e. the GFP molecule missing the hairpin).
Mentions: The structure, stability and folding of GFP have been well studied (Örmo et al., 1996 ▶; Tsien, 1998 ▶; Crameri et al., 1996 ▶). Its relatively simple topology, combined with its utility as a fluorescent reporter when correctly folded (Waldo et al., 1999 ▶; Pédelacq et al., 2006 ▶), has made it an attractive system for reconstitution from separately expressed protein fragments (Cabantous, Terwilliger et al., 2005 ▶). Following such a strategy, by fusing terminal segments of GFP to a crystallization target the resulting construct might be recombined with the remaining complementary fragment of GFP to create a new complex for crystallization. In the context of crystallization strategies, a challenge presented by typical fusion methods is the flexibility introduced at the site of connection between the two protein components; free torsion angles are present where the polypeptide backbone makes its (single) crossing from one natural protein fold to the other. The value of having the polypeptide chain cross twice instead of once between two connected proteins has been demonstrated in experiments in which T4 lysozyme was inserted into a loop of GPCR membrane proteins, giving a construct that yielded well ordered crystals (Rosenbaum et al., 2007 ▶; Cherezov et al., 2007 ▶). The split GFP system [GFP(1–9) + GFP(10–11)] allows a similar advantage. If strands 10 and 11, which ostensibly form a natural hairpin, can be inserted as a long extension into a surface loop of a target protein, then reconstitution with complementary GFP(1–9) should give a tight noncovalent complex with two chain crossings between natural protein folds (Fig. 1 ▶). In practice, rational choices for the points of insertion of strands 10–11 into exposed loops might be based on homology models, where available, or on bioinformatic predictions of loops (Lambert et al., 2002 ▶; Dovidchenko et al., 2008 ▶; Jones, 1999 ▶). Here, we chose a target for crystallization for which the structure was known, in order to test the strategy of loop insertion and crystallization in a favorable case.

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