Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfold ing in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolv ed fluorescence of nine mutants in each of which a single tryptophan was su bstituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W ) exhibited high specific enzymatic activity, and their far-UV CD spectra w ere very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excludin g K1W and Y92W) under folding conditions at 20 degreesC. Like wild-type RNa se A, the various mutants exhibited reversible cooperative thermal unfoldin g transitions at pH 5, with transition temperatures 2.5-11 OC lower than th at of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degreesC, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was m aintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. Fi rst, at temperatures lower than those of the main cooperative transition, l ong-range interactions within the major hydrophobic core are weakened, e.g. , those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta -hairpin motif). The structure of the chain -reversal loop (residues 91-95) relaxes in the same temperature range. Seco nd, the subsequent higher-temperature cooperative unfolding transition is a ssociated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving resi dues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydr ophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the l ocal structural environment of Trp-124 at temperatures slightly above the m ajor cooperative transition. The results shed new light on the thermal unfo lding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.