Peptide backbone circularization enhances antifreeze protein thermostability

Abstract

Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.

Keywords: antifreeze protein; backbone circularization; freezing hysteresis; protein stability; split intein; thermal stability.

Figure 1

Type III AFP backbone cyclization mechanism using protein trans‐splicing. (A) Cartoon depiction of type III AFP (PDB ID:1AME³²) with the modeled backbone circularization loop shown in magenta, blue, and orange, which correspond to N‐extein, C‐extein, and linker colors, respectively, as shown in (B) and (C). Secondary structure elements are colored red (helix), green (β‐strand), and yellow (coil). (B) Schematic of the noncyclized type III AFP construct embedded in the Npu DnaE split‐intein. To facilitate cyclization, the N‐extein and C‐extein moieties are fused to the C‐ and N‐termini, respectively, of the polypeptide to be cyclized. The N‐intein is fused to the C‐terminal end of the N‐extein, and the C‐intein is located at the N‐terminal end of the C‐extein. (C) Products of trans‐splicing by split inteins. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins together via a peptide bond.

Figure 2

MD simulations of wild‐type and three cyclized type III AFP constructs. (A) RSMF values (nm) for the α carbons of wild‐type type III AFP (black line) and cAFPs with GGAA (red), GAA (green), and AA (blue) linkers are plotted against residue number. The 20‐ns simulations were performed on energy‐minimized models of each protein using GROMACS. The solid horizontal line below the graph represents the length of the noncyclized AFP with ice‐binding residues shown by cyan blocks. (B) RMSD values (nm) for all atoms of each AFP construct during the 20‐ns simulations in GROMACS are presented, with averaging of 51 frames to generate a smooth curve. The wild‐type noncyclized type III AFP and cAFP with different linkers are represented by the same colours as in (A). (C) Cartoon models comparing noncyclized AFP to cAFP showing the extein scar (RGKCWE) and the additional flexible linker GAA. Secondary structure elements are colored as in Figure 1.

Figure 3

Purification of circularized type III AFP. (A) Rotary ice‐affinity purification of 1:10 diluted cAFP lysate. (i) Starting ice‐shell; (ii) ice‐shell after 1 h incubation at −1.5°C with cAFP lysate. (B) SDS‐PAGE analysis of ice‐shell purification. Molecular weight standards were electrophoresed in the left‐hand lane. (C) Size‐exclusion purification of ice‐shell‐enriched ice fraction. (D) SDS‐PAGE analysis of size‐exclusion purification fractions of the cAFP with purified noncyclized AFP run alongside for comparison.

Figure 4

Mass analysis of circularized and noncyclized type III AFPs. MALDI‐TOF analysis of cAFP (black line) and noncyclized AFP (blue line). Masses are displayed in Daltons. The peak at 15,683.31 Da is double the mass of the cAFP peak.

Figure 5

NMR analysis of circularized and noncyclized type III AFPs. (A) Overlay of ¹H/¹⁵N‐HSQC spectra of uniformly 13C/¹⁵N‐labeled cAFP (black resonances) and uniformly 15N‐labeled non‐cyclized type III AFP (red resonances). Sequence‐specific backbone resonance assignments are shown as one‐letter amino acid code numbered according to their position in the sequence (Supporting Information Fig. S3). (B) Overlaid strip plots of CBCA(CO)NH (green) and HNCACB (Cα, red and Cβ, blue) of the region linking the C‐ and N‐termini of type III AFP. Dashed lines indicate sequential connectivity of 72R‐73G‐74K‐1C‐2W‐3E.

Figure 6

TH activity of circularized and noncyclized type III AFPs. (A) A molar comparison of TH activity. Black = cAFP; grey = noncyclized AFP. (B) Amount of TH activity after heat treatment at various temperatures. All measurements were performed in triplicate with standard deviation displayed.

Figure 7

¹H–15N HSQC analysis of circularized and non‐cyclized type III AFPs after heating. (A) Spectral overlay of cAFP collected at 25°C after heating at 68°C for 45 min (red resonances) or without heating (black resonances). (B) Spectral overlay of noncyclized AFP collected at 25°C after heating at 68°C for 45 min (red resonances) or without heating (black resonances).