Mutational effects on O(6)-methylguanine-DNA methyltransferase from hyperthermophile: contribution of ion-pair network to protein thermostability.
Ion pairs have been considered to be general stabilizing factors in hyperthermophilic proteins, but the present experimental data cannot fully explain how ion pairs and ion-pair networks contribute to the stability. In this paper, we show experimental evidence that not all of the internal ion pairs contribute to the thermal and thermodynamic stability, using O(6)-methylguanine-DNA methyltransferase from Thermococcus kodakaraensis KOD1 (Tk-MGMT) as a model protein. Of three mutants in which an inter-helical ion pair was disrupted, only one mutant (E93A) was shown to be destabilized. Delta G of E93A was lower by approximately 4 kJ mol(-1) than that of the wild type, and E93A unfolded one order of magnitude faster than did the wild type and other variants. Glu 93 has unique properties in forming an ion-pair network that bridges the N- and C-terminal domains and connects three helices in the protein interior.
Nishikori
Shingo
S
School of Materials Science, Japan Advanced Institute for Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292.
Shiraki
Kentaro
K
Yokota
Kiyonobu
K
Izumikawa
Naoshige
N
Fujiwara
Shinsuke
S
Hashimoto
Hiroshi
H
Imanaka
Tadayuki
T
Takagi
Masahiro
M
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D003198:Computer Simulation; D004795:Enzyme Stability / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D055672:Static Electricity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*; D013816:Thermodynamics; D014162:Transfection; D044366:Transition Temperature
D000596:Amino Acids; D011994:Recombinant Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019791:Guanidine