Mechanism of antifreeze proteins


Mechanism of antifreeze proteins

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) provide survival mechanism for organisms living in subzero environments. AF(G)Ps were found in the blood serums of certain species of fish living in ice-laden marine environments. AFPs also present in certain insects and terrestrial arthropods, and in plants and fungi. It was found that AF(G)Ps are able to lower the freezing point more effectively than any electrolytes that act colligatively, while the melting point and osmolarity are not changed appreciably. AF(G)Ps were also found to inhibit the recrystallization of ice crystals which can damage frozen organisms. Besides the fundamental importance in biological sciences, the study of AF(G)Ps is of broad interest in aquaculture, agriculture and frozen food industries, and has important impact on biomedical research in cold protection of mammalian cells, tissues, and organs.

The goal of our research is to find the antifreeze mechanism including the function, structure, molecular recognition, interaction and dynamics involved in the antifreeze process. This will allow for the subsequent research to find more effective antifreeze materials for biomedical research and other applications.

Experimental setup for observation of ice crystals

Ice lattice and plane of adsorption (left) of Type I AFP (right).

Crystal growth of pure ice: a) seed ice, b) ice crystal grown along the a-axis, c) hexagonal ice crystal formed, d) ice crystal further grown from the edges of hexagonal, e) star like ice crystal formed, and f) ice sheet started to form.

Ice crystal growth in presence of 6 mg/ml type I AFP.

(1) Thermodynamic Mechanism of AFPs for Ice Growth Inhibition

Gibbs-Thomson effect (Kelvin effect)

The binding of AFPs to ice surfaces causes curvatures on ice surface surrounded by the AFP molecules during the growth of ice front. The curvature is energetically less favorable for water molecules to join the ice lattice. The rest of the solution is in a super-cooled condition.

Ice-surface adsorption-enhanced colligative effect of AFPs in ice growth inhibition

AFPs tend to diffuse to water-ice interface to form a Water-AFP-Ice interfacial region due to the decrease in Gibbs energy. The ice growth inhibition arises from the colligative effect of the enhanced AFP concentration in the Water-AFP-Ice interfacial region. Combining the equations of Langmuir adsorption and freezing point depression due to colligative effect leads to the following Equation to relate the mole fraction of water XH2O in the interfacial region and the Thermal hysteresis ∆T of the ice crystal in a super-cooled water.

(Delta)Hfus denotes the heat of fusion of ice, R the gas constant, T0 and Tf the freezing point of pure water and that of water in the Water-AFP-Ice interfacial region, K equilibrium constant for AFP adsorpted in the interfacial region, respectively.

(2) Molecular Mechanism of AFPs for Ice-Growth Inhibition

Structural match of AFPs with ice surface, hydrophobic side chains contacting ice surface and hydrophilic side chains facing to liquid water in the water-AFP-interfacial region contribute to the molecular mechanism of AFP's ice-growth inhibition.

Specific side chain 13C/15N labeled AFPs are designed for using NMR techniques for this study. The NMR techniques include (1) 13C and 15N spin-lattice relaxation NMR; (2) 13C-17O and 15N-17O Rotational Echo-Adiabatic Passage-Double Resonance (REAPDOR) NMR at subzero 0C.

Models of alanine methyl group 13C spin lattice relaxation

Models of methyl group C-C bond continuous rotation (left) and three site rotational
jump (middle) and ice water molecular reorientation (right).

Type I AFP with Alanine Methyl 13C Labels.

Rotational barrier Ea for the methyl C-C rotation/three-site rotational jump.

Rotational barrier Ea for the water molecular reorientation.

REAPDOR (Rotational Echo Adiabatic Passage DOuble Resonance) NMR pulse sequence.

REAPDOR NMR spectra with 6 Tr dipolar dephasing time for (a) AFP i+4, 8 and (b) AFP i+6. The left up spectrum was obtained with dipolar dephasing, and the left down one without dipolar dephasing. The overlap of the two spectra is shown in the right column. The spectral intensity difference shows the methyl 13C proximity to 17O of solid water.
Summary: There were about 10 water molecules closely capping an i +4 or i + 8 methyl group within the range of van der Waals interaction whereas the surrounding water molecules to the i + 6 methyl groups were mobile. The type I AFP side comprising the i + 4 and i + 8 Ala methyl groups interact with the ice surface while side containing i+6 Ala methyl group face to water phase

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