A kinetic method to evaluate the two-state character of solvent-induced protein denaturation.
We present a kinetic method to determine the concentration of native molecules in protein folding transitions. It is based on the observation that frequently native protein molecules unfold slowly when transferred to unfolding conditions, whereas folding intermediates unfold rapidly. The fraction of native molecules in a folding transition can thus be determined by kinetic unfolding assays in a two-step procedure. Aliquots of the protein are first equilibrated at different concentrations of denaturant and then transferred to constant unfolding conditions to determine the amplitude of unfolding. This amplitude is a direct measure for the concentration of native molecules in the sample. The two-state character of a solvent-induced unfolding transition can thus be examined. When the fractional change of a spectral property in a transition follows the decrease in the concentration of the native molecules, as measured by the unfolding assays, then the presence of intermediates that differ from the unfolded protein in this property can be definitely excluded. This test complements the calorimetric test for intermediates in thermal unfolding transitions. By using this method, we show that the NaCl-induced folding transition of the reduced and carboxymethylated form of a variant of ribonuclease T1 is well described by the two-state approximation. In the unfolding of apo-alpha-lactalbumin, the measured profile for the native protein coincides with the fluorescence-detected transition, but not with the transition that is monitored by amide circular dichroism. This confirms that a partially folded intermediate is present in the folding transition of apo-alpha-lactalbumin.
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Laboratorium für Biochemie, Universität Bayreuth, Germany.
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D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D012997:Solvents; D012965:Sodium Chloride; D014508:Urea; D007768:Lactalbumin; D006163:Ribonuclease T1; D019791:Guanidine