Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy.
Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.
Matsuura
Hisae
H
School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan.
Shimotakahara
Sakurako
S
Sakuma
Chiseko
C
Tashiro
Mitsuru
M
Shindo
Heisaburo
H
Mochizuki
Kiwa
K
Yamagishi
Akihiko
A
Kojima
Masaki
M
Takahashi
Kenji
K
D000465:Algorithms; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D006163:Ribonuclease T1