Acid-induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies.
Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies. The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state). In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other. Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states. The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state. A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state. The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate.
Yamasaki
Kazuhiko
K
National Institute of Advanced Industrial Science and Technology, 1-1-1, Higashi, Tsukuba, 305-8566, Japan. k-yamasaki@aist.go.jp
Yamasaki
Tomoko
T
Kanaya
Shigenori
S
Oobatake
Motohisa
M
D000143:Acids / Q000494:pharmacology*; D002942:Circular Dichroism / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000032:analysis*
D000143:Acids; D016914:Ribonuclease H; C078856:ribonuclease HI