Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique

Abstract

The site-directed spin labeling (SDSL) technique was used to examine the antifreeze mechanisms of type-I antifreeze proteins (AFPs). The effects on the growth of seed ice crystals by the spin-label groups attached to different side chains of the AFPs were observed, and the states of water molecules surrounding the spin-label groups were probed via analyses of variable-temperature (VT) dependent electron paramagnetic resonance (EPR) spectra. The first set of experiments revealed the antifreeze activities of the spin-labeled AFPs at the microscopic level, while the second set of experiments displayed those at the molecular level. The experimental results confirmed the putative ice-binding surface (IBS) of type-I AFPs. The VT EPR spectra indicate that type-I AFPs can inhibit the nucleation of seed ice crystals down to ~ - 20 °C in their aqueous solutions. Thus, the present authors believe that AFPs protect organisms from freezing damage in two ways: (1) inhibiting the nucleation of seed ice crystals, and (2) hindering the growth of seed ice crystals once they have formed. The first mechanism should play a more significant role in protecting against freezing damage among organisms living in cold environments. The VT EPR spectra also revealed that liquid-like water molecules existed around the spin-labeled non-ice-binding side chains of the AFPs frozen within the ice matrices, and ice surrounding the spin-label groups melted at subzero temperatures during the heating process. This manuscript concludes with the proposed model of antifreeze mechanisms of AFPs based on the experimental results.

Keywords: Ice crystals; Ice growth inhibition; Ice nucleation inhibition; Site-directed spin labeling; Type-I antifreeze protein; VT EPR.

Fig. 1

Molecular models of a wild-type type-I HPLC6 (side view with ice surface at the bottom), b MSL spin-labeled L12C (side view with ice surface at the bottom), c MSL spin-labeled L23C (side view with ice surface at the bottom), d MSL spin-labeled A20C (top view with ice surface at the bottom); e MSL spin-labeled A11C (top view with ice surface at the bottom), and f MSL spin-labeled A17C (side view with ice surface at the bottom)

Fig. 2

a Photo images of an ice crystal growing in water at 0 °C: (1) seed ice, (2) the ice crystal growing on the prism facets, (3) the ice crystal has formed a hexagonal shape, (4) the ice crystal further growing from the edges of the hexagon, (5) the ice crystal has formed a star-like shape, and (6) the ice crystal burst into the water from a tip of its star-like shape; b photo images of an ice crystal confined by the wild-type type-I AFP at a concentration of 6.0 mg/ml, undergoing decreases in temperature from − 0.09, to − 0.64 and to − 0.65 °C, respectively, from the left to right (the darker circles in the photo images represent air bubbles in the water and the solution)

Fig. 3

Antifreeze activity of the wild-type type-I AFP (black squares), spin-labeled L23C (red discs), spin-labeled L12C (blue, upward-pointing triangles), spin-labeled A20C (purple, downward-pointing triangles), spin-labeled A11C (green diamonds), and spin-labeled A17C (navy blue, leftward-pointing triangles)

Fig. 4

Photo images of ice crystals confined by a MSL-labeled L23C at a concentration of 8.0 mg/ml at − 0.15, − 0.63, and − 0.72 °C, respectively, from left to right; b MSL-labeled L12C at a concentration of 8.0 mg/ml at − 0.08, − 0.34, and − 0.52 °C, respectively, from left to right; c MSL-labeled A20C at a concentration of 8.0 mg/ ml at − 0.10, − 0.65, and − 0.74 °C, respectively, from left to right; d MSL-labeled A11C at a concentration of 8.0 mg/ml at − 0.05, − 0.23, and − 38 °C, respectively, from left to right; and e MSL-labeled A17C at a concentration of 8.0 mg/ml at − 0.01, − 0.02, − 0.02 °C, respectively, from left to right (top), and − 0.02, − 0.01, and − 0.02 °C, respectively, from left to right (down) (the differences in hue were caused by the filters used when the images were taken)

Fig. 5

Experimental (black) and simulated (red) EPR spectra of the MSL spin-labeled HPLC6-mutant solid powders at room temperature

Fig. 6

Experimental (black) and simulated (red) VT EPR spectra of the spin-labeled L12C solution (2.0 mg/ml) undergoing a the cooling process and b the heating process. The temperatures and the correlation times are given in the corresponding legends

Fig. 7

Experimental (black) and simulated (red) VT EPR spectra of the spin-labeled L23C water solution (2.0 mg/ml) undergoing, a the cooling process and b the heating process. The temperatures and correlation times are provided in the corresponding legends

Fig. 8

Experimental and simulated VT EPR spectra of the spin-labeled A20C water solution undergoing, a the cooling process and b the heating process. The temperatures and correlation times are given in the corresponding legends

Fig. 9

Experimental and simulated VT EPR spectra of the spin-labeled HPLC6 A11C water solution (2.0 mg/ml) undergoing, a the cooling process and b the heating process. The temperatures and correlation times are given in the corresponding legends

Fig. 9

Experimental and simulated VT EPR spectra of the spin-labeled HPLC6 A11C water solution (2.0 mg/ml) undergoing, a the cooling process and b the heating process. The temperatures and correlation times are given in the corresponding legends

Fig. 10

Experimental and simulated VT EPR spectra of the spin-labeled A17C solution (2.0 mg/ml) undergoing, a the cooling process and b the heating process. The temperatures and correlation times are provided in the corresponding legends

Fig. 11

Cartoon presentations of the states of water and AFP molecules in the type-I AFP solution at different temperature ranges during the cooling (down) and heating (up) process. The gray color represents hexagonal ice, the blue color represents liquid water, and the orange color represents AFP molecules

Scheme 1

Structure of the MSL spin label and the resulting spin label on the cysteine side chain of a protein