Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.
Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Yoshinori
Y
Matsuura
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Michiyo
M
Takehira
Japan Synchrotron Radiation Research Institute, 1-1-1, Kouto, Sayo, Hyogo 679-5198, Japan.
Yasumasa
Y
Joti
Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Kyoko
K
Ogasahara
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Tomoyuki
T
Tanaka
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Naoko
N
Ono
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Naoki
N
Kunishima
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Katsuhide
K
Yutani
D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D013816:Thermodynamics
C094846:CutA protein, E coli; D029968:Escherichia coli Proteins; D050505:Mutant Proteins