The formation of natural gas hydrates presents significant economic and safety challenges to the petroleum and gas industry, necessitating the development of effective prevention strategies. This study investigates an environmentally sustainable Tenebrio molitor antifreeze protein (TmAFP) modified to be a potential kinetic hydrate inhibitor. The aim of this study was to enhance the inhibitory activity of TmAFP by systematically substituting threonine (Thr) residues with glycine (Gly), alanine (Ala), or serine (Ser) at positions 29, 39, and 53. The Ala mutant demonstrated superior inhibition of hydrate formation, attributed to its optimized spatial conformation and enhanced hydrophobic interactions, followed by the Gly and Ser mutants. The wild-type TmAFP showed limited efficacy. The radial distribution function (RDF) analysis indicated that the mutations facilitated a better accommodation of adjacent residues within the hydrate crystal structure by adjusting the distance between Thri and Thri+2 to closely match the second peak in the RDF of methane molecules at 6.4 Å. The potential of mean force (PMF) calculations revealed that the Ala and Ser mutants exhibited enhanced interactions with hydrate cages, with PMF values of -0.73 and -0.71 kJ/mol, respectively, compared to the Gly mutant, which had a PMF value of 1.46 kJ/mol. By identifying the optimal mutation combination (T29 39 53A) to significantly increase the potency of TmAFP, this study provides a fundamental basis for the further development of hydrate inhibition strategies.
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