【摘要】 嗜热古菌蛋白Ssh10b突变体[P62A]Ssh10b具有良好的热稳定性.[P62A]Ssh10b的结构测定结果显示,其α2螺旋上残基K48和D51之间形成了一对盐键,而且,突变D51将影响蛋白的热稳定性.为了探索D51的突变对蛋白热稳定性的影响,构建了突变体[D51N/P62A]Ssh10b的质粒,并获得了高纯度的15N和13C双标记[D51N/P62A]Ssh10b.通过对异核三共振NMR实验数据的解析,完成了对[D51N/P62A]Ssh10b的主链共振近乎完全的指认.比较突变以及未突变蛋白质的主链1HN和15N化学位移,在[P62A]Ssh10b的结构基础上进行分析发现,D51N突变显著地影响了α2螺旋骨架构象,并进一步影响到古菌Lβ2α2loop区域、β4的N端区域、Lβ3β4loop的C端区域以及β3与Lβ3β4loop的交界区域.结果表明,由于D51N突变破坏了α2螺旋上K48和D51之间的盐键,影响了[D51N/P62A]Ssh10b的α2螺旋构象,并影响到蛋白的其它相关部位的局部构象,说明[P62A]Ssh10b的高热稳定性可能与其溶液构象密切相关.为进一步运用NMR研究[D51N/P62A]Ssh10b分子结构特性与耐热机制间的关系奠定了基础.更多还原

【Abstract】 The P62A mutant hyperthermophilic archaeal protein Ssh10b,[P62A]Ssh10b,is highly thermostable.The structure determination shows that a salt-bridge K48-D51 is formed in helix α2,and mutation of D51 can influence the thermostability of protein.In order to understand the effect of mutation on the stability of protein,we constructed the D51N-mutant [P62A]Ssh10b plasmid,and obtained the 15N and 13C double-labeled [D51N/P62A]Ssh10b protein with a high purity.The experimental data of triple-resonance NMR experiments provided nearly complete backbone resonance assignments.Comparing the main chain 1HN and 15N chemical shifts for [P62A]Ssh10b with its mutant variant and analyzing the chemical shift differences on the basis of [P62A] Ssh10b structure,it was found that D51N mutation influenced the backbone conformation of helix α2.The further effect was observed in the structural region consisting of Loop Lβ2α2,the N-terminal of strand β4,and the C-terminal of loop Lβ3β4,and in the region around loop Lβ3β4 as well.This revealed that D51N mutation disrupted the salt bridge of K48-D51 on helix α2 and influenced not only the backbone conformation of helix α2 but also the local conformations of other structural regions in the protein.Therefore,the high thermostability of [P62A]Ssh10b may be correlated closely to its tertiary conformation.更多还原