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 ¹H^N 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 L_β2α2, the N-terminal of strand β4, and the C-terminal of loop L_β3β4, and in the region around loop L_β3β4 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.

Key words: D51N mutant, [D51N/P62A]Ssh10b, NMR, Backbone resonance assignment, Chemical shift comparison