New structural insights into the refolding of ribonuclease T1 as seen by time-resolved fourier-transform infrared spectroscopy

Abstract

To get new structural insights into different phases of the renaturation of ribonuclease T1 (RNase T1), the refolding of the thermally unfolded protein was initiated by rapid temperature jumps and detected by time- resolved Fourier-transform infrared spectroscopy. The characteristic spectral changes monitoring the formation of secondary structure and tertiary contacts were followed on a time scale of 10-3 to 103 seconds permitting the characterization of medium and slow folding reactions. Additionally, structural information on the folding events that occurred within the experimental dead time was indirectly accessed by comparative analysis of kinetic and steady-state refolding data. At slightly destabilizing refolding temperatures of 45°C, which is close to the unfolding transition region, no specific secondary or tertiary structure is formed within 180 ms. After this delay all infrared markers bands diagnostic for individual structural elements indicate a strongly cooperative and relatively fast folding, which is not complicated by the accumulation of intermediates. At strongly native folding temperatures of 20°C, a folding species of RNase T1 is detected within the dead time, which already possesses significant amounts of antiparallel β-sheets, turn structures, and to some degree tertiary contacts. The early formed secondary structure is supposed to comprise the core region of the five-stranded β-sheet. Despite these nativelike characteristics the subsequent refolding events are strongly heterogeneous and slow. The refolding under strongly native conditions is completed by an extremely slow formation or rearrangement of a locally restricted β-sheet region accompanied by the further consolidation of turns and denser backbone packing. It is proposed that these late events comprise the final packing of strand 1 (residues 40-42) of the five-stranded β-sheet against the rest of this β-sheet system within an otherwise nativelike environment. This conclusion was supported by the comparison of refolding of RNase T1 and its variant W59Y RNase T1 that enabled the assignment of these very late events to the trans → cis isomerization reaction of the prolyl peptide bond preceding Pro-39.