Biophysical Journal 66: 40-45 (1994)
© 1994 the Biophysical Society

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

Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul.

ABSTRACT

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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