Mutation studies on transmembrane protein sulphate permease for enhanced biohydrogen production in Chlamydomonas reinhardtii: a molecular dynamics simulation approach

Fossil fuel reserves are rapidly depleting, necessitating the need to find a promising alternative. Hydrogen is a clean and promising energy source with a significant energy yield and water as the only byproduct leading many to view it as a viable source of renewable energy. Despite traditional assessments for the techno-economic feasibility of biohydrogen not supporting its practical implementation, biologically produced hydrogen remains a more environmentally friendly and an efficient energy source. Recent studies have reported that low levels of sulphate permease (SulP) is said to increase hydrogen production. Mutation studies were carried out and molecular docking was performed for wild type and mutated sulphate permease with ligands that dampen its activity as reported in literature. Effect of mutation on SULP1 gene in Chlamydomonas reinhardtii was assessed using PredictSNP, I-Mutant 2.0, and SIFT. Sulphate permease being a transmembrane protein, CHARMM-GUI membrane builder module was used for solvent equilibration, lipid bilayer generation and proteins. Protein structures were harmonically restrained to maintain their initial positions and allow the solvent to equilibrate effectively. Molecular simulations were performed for wild & mutant types with ligands and assessed for RMSD, RMSF, intra- and inter-hydrogen bond interactions, Rg and SASA till 200 ns using GROMACs v.2016. The MMPBSA values were found to be −29.392 +/− 11.753 kJ/mol WT (Wild type) and −34.080 +/− 10.837 kJ/mol MT (Mutant type). Significant structural modification was inferred through the mutation approach.化石燃料储量正快速枯竭，亟需探寻极具潜力的替代能源。氢能是一种清洁且极具发展前景的能源，其能量产率可观，唯一副产物仅为水，因此被诸多研究者视为可行的可再生能源方案。尽管针对生物氢能的技术经济可行性传统评估结果并不支持其实际落地，但生物制氢仍是一种更为环境友好且高效的能源。近期研究表明，低表达的硫酸盐通透酶（sulphate permease, SulP）可提升氢气产率。本研究开展了突变实验，并针对野生型与突变型硫酸盐通透酶，与文献中报道的可抑制其活性的配体进行了分子对接操作。针对莱茵衣藻（Chlamydomonas reinhardtii）SULP1基因的突变效应，通过PredictSNP、I-Mutant 2.0及SIFT工具进行了评估。鉴于硫酸盐通透酶属于跨膜蛋白，本研究采用CHARMM-GUI膜构建模块，完成溶剂平衡、脂质双层构建及蛋白预处理。对蛋白结构施加谐波约束，以维持其初始构象，确保溶剂能够充分平衡。采用GROMACS v.2016软件，针对结合配体的野生型与突变型蛋白开展了200 ns的分子动力学模拟，并对其均方根偏差（Root Mean Square Deviation, RMSD）、均方根波动（Root Mean Square Fluctuation, RMSF）、分子内与分子间氢键相互作用、回转半径（Radius of Gyration, Rg）及溶剂可及表面积（Solvent Accessible Surface Area, SASA）进行了分析评估。经分子力学-泊松玻尔兹曼表面积法（Molecular Mechanics/Poisson-Boltzmann Surface Area, MMPBSA）分析，野生型（WT，Wild type）的结合自由能为-29.392 ±11.753 kJ/mol，突变型（MT，Mutant type）为-34.080 ±10.837 kJ/mol。研究结果表明，该突变手段可引发蛋白结构的显著改变。