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Hisae Matsuura, Sakurako Shimotakahara, Chiseko Sakuma, Mitsuru Tashiro, Heisaburo Shindo, Kiwa Mochizuki, Akihiko Yamagishi, Masaki Kojima, Kenji Takahashi
Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy
Keywords: nuclear magnetic resonance, residual structure, ribonuclease T1, singular value decomposition, thermal unfolding
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 ?-helix and two ?-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°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 ?-strands (B3, B4, and B5 in ?-sheet B). These ?-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°C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 385, 12/2004
Pages: 1157 - 1164