Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

Abstract

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Figure 1.

The refolding and unfolding kinetics of 30 small proteins and isolated protein domains. All but one fold with rates that are well-fitted (r² > 0.97) by linear-arm chevrons (equation 2) and appear to represent two-state folding under these conditions (as judged by the equivalence of the folding free energy as determined from kinetic and equilibrium chemical melts) (Table 1). The protein U1A exhibits significantly curved folding and unfolding arms under the conditions employed here (and other conditions)) (Otzen et al. 1999). Fitting the apparently linear region of the U1A chevron (r²= 0.994) produces an estimated ln(k_f) in water of 7.68 ± 0.18; fitting all of the data to a second order polynomial chevron (r² = 0.9995) produces the significantly lower estimate ln(k_f) = 4.62 ± 0.05. Several other proteins appear to exhibit curvature in their unfolding arms. But because these data are also well-fitted by equation 2, this curvature was ignored in the illustrated fits and in the data reported in Table 1. Note that the X- and Y-axis ranges vary significantly from plot to plot in this figure.

Figure 1.

The refolding and unfolding kinetics of 30 small proteins and isolated protein domains. All but one fold with rates that are well-fitted (r² > 0.97) by linear-arm chevrons (equation 2) and appear to represent two-state folding under these conditions (as judged by the equivalence of the folding free energy as determined from kinetic and equilibrium chemical melts) (Table 1). The protein U1A exhibits significantly curved folding and unfolding arms under the conditions employed here (and other conditions)) (Otzen et al. 1999). Fitting the apparently linear region of the U1A chevron (r²= 0.994) produces an estimated ln(k_f) in water of 7.68 ± 0.18; fitting all of the data to a second order polynomial chevron (r² = 0.9995) produces the significantly lower estimate ln(k_f) = 4.62 ± 0.05. Several other proteins appear to exhibit curvature in their unfolding arms. But because these data are also well-fitted by equation 2, this curvature was ignored in the illustrated fits and in the data reported in Table 1. Note that the X- and Y-axis ranges vary significantly from plot to plot in this figure.

Figure 1.

The refolding and unfolding kinetics of 30 small proteins and isolated protein domains. All but one fold with rates that are well-fitted (r² > 0.97) by linear-arm chevrons (equation 2) and appear to represent two-state folding under these conditions (as judged by the equivalence of the folding free energy as determined from kinetic and equilibrium chemical melts) (Table 1). The protein U1A exhibits significantly curved folding and unfolding arms under the conditions employed here (and other conditions)) (Otzen et al. 1999). Fitting the apparently linear region of the U1A chevron (r²= 0.994) produces an estimated ln(k_f) in water of 7.68 ± 0.18; fitting all of the data to a second order polynomial chevron (r² = 0.9995) produces the significantly lower estimate ln(k_f) = 4.62 ± 0.05. Several other proteins appear to exhibit curvature in their unfolding arms. But because these data are also well-fitted by equation 2, this curvature was ignored in the illustrated fits and in the data reported in Table 1. Note that the X- and Y-axis ranges vary significantly from plot to plot in this figure.

Figure 1.

The refolding and unfolding kinetics of 30 small proteins and isolated protein domains. All but one fold with rates that are well-fitted (r² > 0.97) by linear-arm chevrons (equation 2) and appear to represent two-state folding under these conditions (as judged by the equivalence of the folding free energy as determined from kinetic and equilibrium chemical melts) (Table 1). The protein U1A exhibits significantly curved folding and unfolding arms under the conditions employed here (and other conditions)) (Otzen et al. 1999). Fitting the apparently linear region of the U1A chevron (r²= 0.994) produces an estimated ln(k_f) in water of 7.68 ± 0.18; fitting all of the data to a second order polynomial chevron (r² = 0.9995) produces the significantly lower estimate ln(k_f) = 4.62 ± 0.05. Several other proteins appear to exhibit curvature in their unfolding arms. But because these data are also well-fitted by equation 2, this curvature was ignored in the illustrated fits and in the data reported in Table 1. Note that the X- and Y-axis ranges vary significantly from plot to plot in this figure.

Figure 1.

The refolding and unfolding kinetics of 30 small proteins and isolated protein domains. All but one fold with rates that are well-fitted (r² > 0.97) by linear-arm chevrons (equation 2) and appear to represent two-state folding under these conditions (as judged by the equivalence of the folding free energy as determined from kinetic and equilibrium chemical melts) (Table 1). The protein U1A exhibits significantly curved folding and unfolding arms under the conditions employed here (and other conditions)) (Otzen et al. 1999). Fitting the apparently linear region of the U1A chevron (r²= 0.994) produces an estimated ln(k_f) in water of 7.68 ± 0.18; fitting all of the data to a second order polynomial chevron (r² = 0.9995) produces the significantly lower estimate ln(k_f) = 4.62 ± 0.05. Several other proteins appear to exhibit curvature in their unfolding arms. But because these data are also well-fitted by equation 2, this curvature was ignored in the illustrated fits and in the data reported in Table 1. Note that the X- and Y-axis ranges vary significantly from plot to plot in this figure.