Kinetic Evidence of Microscopic States in Protein Folding

Hueih M. Chen, Vladislav S. Markin, Tian Yow Tsong

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21 Scopus citations

Abstract

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3⇌D2⇌D1⇌N0, in which No is the native state and D's are the three substates of the denatured form [Chen, M.M., You, J. L, Markin, V.S., & Tseng, T.Y. (1990) J. Mol. Biol. 220, 771-778; Chen, H.M., Markin, V.S., & Tseng, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the singleand the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25 °C, log τ1 1(for the D1⇌ N0step) and log τ2 -1(for the D2⇌ D1step) vs pH show a [Formula Omiitted]shaped dependence on the final pH, with minimal values (τ1 1of 0.56 s-1and τ1 2of around pH 3.9. The third relaxation τ3(for the D3⇌ D2step, 35 s) was independent of pH in the range 3.4-8.5. The [Formula Omiitted]-shaped dependence on pH of log τ1 1and log τ1 2cannot be reproduced by the above but can be accounted for if each of N0, D1, and D2is composed of many microscopic states in rapid equilibrium. These microscopic states are designated as two subpopulations, αi(i = 0, 1, and 2), from which unfolding can take place, and ±i, (i = 1, 2, and 3), from which folding can take place. Analysis of kinetic data indicates that α1= 1 and α2= 1 for the whole pH range. However, β = 1 only for pH>5.5 and decreases to 0 for pH<3 (pKaaround 4.5), and β2 = 1 only for pH<2 and decreases to small values for pH>3 (pKaaround 1.2). Analysis also shows that equilibrium unfolding of the protein is triggered by the absorption of 2.6 ± 0.3 protons, of which 0.8 ± 0.3 occurs in the No to Di step and 1.7 ± 0.3 occurs in the D1to D2step. We conclude that acidic unfolding can take place from the whole population of microscopic states while folding at pH 7 can take place only through those subpopulations which are folding-permitting. pH-dependent equilibrium transitions of all kinetically distinctive intermediates in the folding pathway are also determined and shown to be consistent with the above interpretation.

Original languageEnglish (US)
Pages (from-to)12369-12375
Number of pages7
JournalBiochemistry
Volume31
Issue number49
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry

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