Absence of stable intermediates on the folding pathway of barnase

Abstract

Barnase is one of the few protein models that has been studied extensively for protein folding. Previous studies led to the conclusion that barnase folds through a very stable submillisecond intermediate ( approximately 3 kcal/mol). The structure of this intermediate was characterized intensively by using a protein engineering approach. This intermediate has now been reexamined with three direct and independent methods. (i) Hydrogen exchange experiments show very small protection factors ( approximately 2) for the putative intermediate, indicating a stability of approximately 0.0 kcal/mol. (ii) Denaturant-dependent unfolding of the putative intermediate is noncooperative and indicates a stability less than 0.0 kcal/mol. (iii) The logarithm of the unfolding rate constant of native barnase vs. denaturant concentrations is not linear. Together with the measured rate ("I" to N), this nonlinear behavior accounts for almost all of the protein stability, leaving only about 0.3 kcal/mol that could be attributed to the rapidly formed intermediate. Other observations previously interpreted to support the presence of an intermediate are now known to have alternative explanations. These results cast doubts on the previous conclusions on the nature of the early folding state in barnase and therefore should have important implications in understanding the early folding events of barnase and other proteins in general.

Figure 1

Chevron curves of wild-type barnase at 25°C and pH 6.3 (solid circles) and pH 7.23 in the presence of 0.1 M Na₂SO₄ (open circles). The solid curve is the best fit of the observed rate constants to a three-state model with Eqs. 1–3. It yields the following values: K_IU = 0.009; m_IU = 4.05 M⁻¹; k_IN = 11.9 s⁻¹; m_IN = 2.63 M⁻¹; k_NI = 8.6 × 10⁻⁶ s⁻¹; m_NI = 2.80 M⁻¹; m_NU (sum of m_IU, m_IN, and m_NI) = 9.49 M⁻¹; and m_IU/m_NU = 0.43. The dashed line illustrates the linear extrapolation of unfolding rate constant log k_u. It should be noted that the k_u in water, obtained with guanidinium chloride (GdmCl) and linear extrapolation, is 10 times smaller than the value obtained with urea (1).

Figure 2

HX protection factors in the early folding state. The filled bars are the P_f values of the 13 amide protons. The asterisks indicate the averaged P_f measured at both pH 6.3 and 7.23. Others in open bars are at pH 7.23.

Figure 3

Noncooperative unfolding of the postulated intermediate. Kinetic traces generated by the stopped-flow CD at 0.94 M urea (A) and fluorescence in water (B) at pH 6.3 and 25°C. The values at 10 ms of folding as indicated by the arrows at different urea concentrations were plotted in C (solid circles) and D (solid circles). The global unfolding of barnase is shown with the dotted line. The curved solid line is the fitting curve with a two-state model with parameters for both pretransition and posttransition baselines (see Materials and Methods). The dashed line represents the posttransition baseline. Mdeg, millidegree.

Figure 4

Downward deviation of the log k_u at 30°C and pD 6.8 (pD_read + 0.4) in D₂O. The open and filled squares are rate constants measured by using stopped-flow fluorescence under the conditions used in the native-state HX experiments (9). The open and filled circles represent the HX rate constants of the slowly exchanging amide protons measured at GdmCl concentrations from 0.0 to 0.51 M and from 0.73 M to 1.20 M, respectively (9). The convergence of the exchange rate constants of the 13 amide protons at above 0.51 M GdmCl is indicative of EX1 exchange mechanism. The solid straight line represents the linear fitting of the filled circles with a slope of 2.425 and an intercept of −5.924. The dashed line is the linear fitting to the log k_u at higher GdmCl concentrations.