突变对嗜热古菌蛋白[P62A]Ssh10b主链构象影响的核磁共振研究

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、Lβ3β4 loop的C端区域以及β3与Lβ3β4 loop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.

NMR Study of Influences of D51N Mutation on Backbone Conformation of Hyperthermophilic Archaeal [P62A]Ssh10b

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.