Comparison of kinetics of formation of helices and hydrophobic core during the folding of staphylococcal nuclease from acid.

摘要

Our previous kinetic study of the acid and base-induced folding/unfolding transitions of staphylococcal nuclease (SNase) has monitored Trp-140 fluorescence. Trp-140 is located near the flexible COOH terminus and whether or not its fluorescence reflects the overall conformation of the protein has yet to be established. Here we show that the fluorescence intensity of Try-140 correlated closely with the thermal stability (i.e., the calorimetric enthalpy, delta Hcal, of unfolding) of the protein in the pH range 7 to 2.5, confirming that it is a good measure of the overall protein structure. Circular dichroism (CD) at 222 nm, which reflects the helical content of the protein molecule, was used to follow the same folding/unfolding transition in order to compare kinetics of the helix formation and of the appearance of the hydrophobic core. In addition to the three kinetic phases reported earlier with the fluorescence detection, there were a rapid reaction (completed within the 25 ms mixing time of the instrument), which comprised 15% of the signal, and a very slow reaction (time constant > 300 s), which comprised 19% of the signal. With the fluorescence detection for the folding from acid, only 5% of the signal occurred in the rapid phase and there was no reaction slower than 300 s. By comparing kinetics of folding at pH 7 by the CD and fluorescence detection methods, we concluded that: (a) Roughly 15% of the helix content of SNase accumulated before significant changes in the hydrophobic environment (< 5%) of Trp-140 could be detected. The rapid appearance of CD signal reminiscent of helix formation within 25ms would be consistent with the framework model of protein folding. Note, however, that, 15% of the 22% helix content of the protein amounts to an equivalent of fewer than 5 amino acid residues. (b) For the time-resolved signal between 2 ms and 300s, kinetics measured by both properties are consistent with the sequential model, D4 = D3 = D2 = D1 = No (the four Ds are the four substates of the denatured protein and No is the native state). The major folding step by both signals is the D1 to No transition, which gave approximately a 50% change in fluorescence and CD and had a time constant of 160 ms at 25 degree C, pH7.0. (c) The slow phase with the CD signal (>300 s), which is insensitive to Trp-140 fluorescence, has been assigned to be the cis/trans isomerization of Pro-1 17 by other studies. (d) Kinetics in the unfolding direction are consistent with the above interpretation.