Abstract
Antifreeze protein (AFP) has a unique function of reducing solution freezing temperature to protect organisms from ice damage. However, its functional mechanism is not well understood. An intriguing question concerning AFP function is how the high selectivity for ice ligand is achieved in the presence of free water of much higher concentration which likely imposes a large kinetic barrier for protein-ice recognition. In this study, we explore this question by investigating the property of the ice binding surface of an antifreeze protein using NMR spectroscopy. An investigation of the temperature gradient of amide proton chemical shift and its correlation with chemical shift deviation from random coil was performed for CfAFP-501, a hyperactive insect AFP. A good correlation between the two parameters was observed for one of the two Thr rows on the ice binding surface. A significant temperature-dependent protein-solvent interaction is found to be the most probable origin for this correlation, which is consistent with a scenario of hydrophobic hydration on the ice binding surface. In accordance with this finding, rotational correlation time analyses combined with relaxation dispersion measurements reveals a weak dimer formation through ice binding surface at room temperature and a population shift of dimer to monomer at low temperature, suggesting hydrophobic effect involved in dimer formation and hence hydrophobic hydration on the ice binding surface of the protein. Our finding of hydrophobic hydration on the ice binding surface provides a test for existing simulation studies. The occurrence of hydrophobic hydration on the ice binding surface is likely unnecessary for enhancing protein-ice binding affinity which is achieved by a tight H-bonding network. Subsequently, we speculate that the hydrophobic hydration occurring on the ice binding surface plays a role in facilitating protein-ice recognition by lowering the kinetic barrier as suggested by some simulation studies.
Highlights
Antifreeze protein (AFP), discovered decades ago [1], is widely exploited by a variety of organisms to deal with the harsh condition at freezing temperatures [2,3]
The NMR chemical shift is sensitive to the chemical environment of the observing nuclei and its temperature gradient contains information on temperature associated structural or dynamic changes [19]
Most residues fall in the range of 22.061.4 ppb/uC, which is typical for H-bonded exchange-protected amide protons according to statistical analysis of protein and peptides [20]
Summary
Antifreeze protein (AFP), discovered decades ago [1], is widely exploited by a variety of organisms to deal with the harsh condition at freezing temperatures [2,3]. For moderately active AFP, such as type I fish AFP, sufficient ice binding affinity apparently can be contributed by either Hbonding or hydrophobic interactions as suggested by various mutagenesis studies [6,7,8]. It is hard to tackle this question experimentally, whereas various simulation studies pointed out the occurrence of hydrophobic hydration on icebinding surface, and/or suggested that the interfacial water is mediating protein-ice recognition [2,11,12,13,14,15,16]. We explore this question by investigating the structural/dynamic property of the ice-binding surface of an antifreeze protein using NMR spectroscopy
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