The influence of a type I antifreeze protein and its mutants on methane hydrate adsorption-inhibition: A molecular...

The formation of methane hydrates in oil and gas pipelines poses significant challenges to flow assurance, driving the urgent need for efficient and environmentally friendly kinetic hydrate inhibitors. The moderately active type I winter flounder antifreeze protein (wfAFP) exerts its ability to inhibit methane hydrate growth by binding to methane hydrate cages via specific amino acids [Thr-(i), Ala-(i+4), Ala-(i+7), where i = 2, 13, and 24]. However, the design strategy to enhance its methane hydrate growth inhibitory activity remains incompletely elucidated. In this study, we designed modifications to improve wfAFP's methane hydrate growth inhibitory capacity based on key positions. The results demonstrate that the simultaneous mutation of Ala to Thr at the Ala-(i+4) and Ala-(i+7) positions (i = 2, 13, and 24) significantly enhances its ability to inhibit methane hydrate growth. Further analysis reveals that this system exhibits the lowest methane hydrate content and forms the highest number of hydrogen bonds with surrounding water molecules (with the longest lifetime), and the entire protein adopts a stable adsorption conformation parallel to the methane hydrate surface. The hydroxyl groups of threonine residues maintain the hydrogen bond network during binding to methane hydrates, while the methyl groups stabilize the hydrophobic embedding structure within hydrate cages. These findings provide a method for enhancing the methane hydrate growth inhibitory activity of moderately active AFPs and offer theoretical support for designing efficient and environmentally friendly hydrate inhibitors.