Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein.
Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.
Takano
Kazufumi
K
Department of Material and Life Science, Osaka University, Yamadaoka, Suita 565-0871, Japan. ktakano@mls.eng.osaka-u.ac.jp
Higashi
Ryogo
R
Okada
Jun
J
Mukaiyama
Atsushi
A
Tadokoro
Takashi
T
Koga
Yuichi
Y
Kanaya
Shigenori
S
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D007700:Kinetics; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*; D016415:Sequence Alignment; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology
D011392:Proline; C091139:ribonuclease HII; D016914:Ribonuclease H