Distinct mechanisms of methane hydrate growth inhibition by ice-binding and non-ice-binding sites in a hyperactive insect antifreeze protein

• Non-IBS significantly inhibits the growth of methane hydrate through the adsorption of specific amino acid residues on the surface of methane hydrate, especially HIS33, PHE59, and TYR71. • The IBS mainly relies on perturbation instead of direct adsorption to obtain the property of inhibiting the growth of methane hydrate. • Both the IBS and non-IBS have inhibitory effects on methane hydrate. It is challenging for these residues on the IBS to generate deformation to form a binding with the nascent hydrate cage. The inhibitory mechanisms of organism-evolved antifreeze proteins (AFPs) on methane hydrate growth have been extensively investigated. However, comprehensive understanding of the mechanistic differences between ice-binding site (IBS) and non-ice-binding site (non-IBS) of AFPs remains fragmentary. This study systematically examines the atomic-level interactions of IBS and non-IBS in a hyperactive insect antifreeze protein throughout the entire inhibition process of methane hydrate (001) plane, through comparative analysis with a non-antifreeze protein control. The results demonstrate the non-IBS mainly inhibits the growth of methane hydrate by adsorbing specific amino acid residues on its surface. However, for the IBS, the distances between amino acids do not match the neighboring cages of methane hydrate. Moreover, on the β-sheet, these residues are not influenced by a considerable number of disulphides and hydrogen bonds, and it is challenging to generate deformation to form a binding with the nascent hydrate cage. It is concluded that the IBS mainly inhibits the growth of methane hydrate through perturbation effects. This study reveals the adsorption mechanism of the non-IBS and the perturbation inhibition mechanism of the IBS. The results are beneficial for the design of antifreeze proteins and bioinspired materials with superior performance for inhibiting the growth of methane hydrate.