A straight path to circular proteins

Abstract

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.

FIGURE 1.

Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).

FIGURE 2.

Cyclization of Cre recombinase. A, G-Cre-LPETG-His₆ (50 μm) was incubated with sortase A (50 μm) in the presence or absence of fluorescent GGG-TMR (10 mm) in sortase reaction buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm CaCl₂) for 21.5 h at 37 °C. SDS-PAGE revealed the expected C-terminal transpeptidation product when GGG-TMR was included, whereas omission of the triglycine nucleophile resulted in clean conversion to a unique protein species with a lower apparent molecular weight. Schematic representations to the right of the gel indicate the topology of the protein species produced by transpeptidation. B, ESI-MS of linear G-Cre-LPETG-His₆ and circular Cre formed by intramolecular transpeptidation. C, MS/MS spectrum of a tryptic fragment of circular Cre showing the ligation of the N-terminal residues (GEFAPK) to the C-terminal LPET motif. Expected masses for y and b ions are listed above and below the peptide sequence. Ions that were positively identified in the MS/MS spectrum are highlighted in blue or red. Only the most prominent daughter ions have been labeled in the MS/MS spectrum.

FIGURE 3.

Cyclization of Cre is reversible. A, G-Cre-LPETG-His₆ (50 μm) was circularized by treatment with sortase A (50 μm) in sortase reaction buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm CaCl₂) for 21.5 h at 37 °C (lane 1). This reaction mixture was then treated with 10 mm GGG-TMR (lane 3) or simply incubated for an additional 24 h at 37 °C (lane 2). All reactions were analyzed by SDS-PAGE with visualization by Coomassie staining or in-gel fluorescence. For comparison, a C-terminal labeling reaction performed using 10 mm GGG-TMR without prior cyclization of the Cre substrate (lane 4) and a sample of linear G-Cre-LPETG-His₆ incubated without sortase A or nucleophile (lane 5) are included. B, molecular model of Cre recombinase monomer (generated from PDB code 1kbu) (29) showing the proximity relationship between the N and C termini. The N-terminal glycine residue is highlighted in red, and the C-terminal LPET residues are shown in green.

FIGURE 4.

Circularization of eGFP. A, molecular model of eGFP (generated from PDB code 1gfl) (31) showing the proximity relationship between the N and C termini. The N-terminal glycine residue is highlighted in red, and the C-terminal LPET residues are shown in green. B, G₅-eGFP-LPETG-His₆ (50 μm) was circularized by treatment with sortase A (50 μm) in sortase reaction buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm CaCl₂) for 24 h at 37 °C. Schematic representations to the right of the gel indicate the topology of the protein species produced by transpeptidation. C, circular eGFP (C) recovers fluorescence more rapidly than linear G₅-eGFP-LPETG-His₆ (L) following thermal denaturation for 5 min at 90 °C. SDS-PAGE analysis confirmed the purity and concentration of the circular eGFP (C) and linear G₅-eGFP-LPETG-His₆ (L) samples used for refolding.

FIGURE 5.

UCHL3 with an internally positioned LPETG motif is circularized by sortase A. A, molecular model of human UCHL3 (generated from PDB code 1xd3) (30) showing the active site crossover loop bearing an LPETG substitution (LPET residues shown in green and Gly residue shown in red). The N-terminal glycine residue that serves as the nucleophile for intramolecular transpeptidation is highlighted in red. B, UCHL3 (30 μm) bearing an LPETG substitution in the active site crossover loop was incubated with sortase A (150 μm) in the absence or presence of GGG nucleophile (90 mm) in sortase reaction buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm CaCl₂) for the indicated times at 37 °C and analyzed by SDS-PAGE, followed by Coomassie staining. Schematic representations to the right of the gel indicate the topology of the protein species produced by transpeptidation.

FIGURE 6.

Positions of the N and C termini in the p97 hexamer are suitable for intermolecular cross-linking through sortase-catalyzed transpeptidation. A, molecular model of p97 trimer (generated from PDB code 3cf1) (28) showing the relative position of p97 monomers in the hexameric ring. The visible N and C termini from the published p97 trimer structure are indicated in red and green, respectively. B, molecular model of G-His₆-p97-LPSTG-XX showing the proximity relationship between N and C termini in adjacent p97 monomers. N- and C-terminal residues not visible in the published crystal structure have been modeled onto the existing structure. N-terminal glycine residues are shown in red, and the C-terminal LPST residues are shown in green. For clarity, the C-terminal domains of the outer monomers are hidden, as is the N-terminal domain of the central monomer. C, G-His₆-p97-LPSTG-XX (1.5 mg/ml) was incubated with sortase A (30 μm) in sortase reaction buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm CaCl₂) for the times indicated at 37 °C. After 22 h, diglycine (GG) was added (100 mm final concentration), resulting in disappearance of the covalent oligomers (lane 5). For comparison, a control reaction containing 100 mm diglycine peptide from the outset of the experiment is shown in lane 6.