A stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [P62A]Ssh10b.
The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.
Fang
Xianyang
X
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Cui
Qiu
Q
Tong
Yufeng
Y
Feng
Yingang
Y
Shan
Lu
L
Huang
Li
L
Wang
Jinfeng
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D019843:Archaeal Proteins; D019641:DNA, Archaeal