Thermal unfolding of ribonuclease T1 studied by multidimensional NMR spectroscopy

Thermal unfolding of ribonculease (RNase) T1 was studied by H-1 nuclear Overhauser enhancement spectroscopy (NOESY) and H-1-N-15 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 54.0degreesC 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 56degreesC. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.