Role of charged residues in stabilization of Pyrococcus horikoshii CutA1, which has a denaturation temperature of nearly 150 °C.
The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.
RIKEN SPring-8 Center, RIKEN Harima Institute, Sayo, Hyogo, Japan.
Yoshinori
Y
Matsuura
Michiyo
M
Takehira
Masahide
M
Sawano
Kyoko
K
Ogasahara
Tomoyuki
T
Tanaka
Hitoshi
H
Yamamoto
Naoki
N
Kunishima
Etsuko
E
Katoh
Katsuhide
K
Yutani
D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D013816:Thermodynamics
D019843:Archaeal Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid