Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues

Abstract

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition.

Figure 1

Equilibrium urea-induced unfolding of WT apomyoglobin and some of its representative mutant forms at pH 6.2: (a), as detected by molar ellipticity at 222 nm ([θ]₂₂₂); (b), as detected by changing intensity of tryptophan fluorescence at 335 nm (I₃₃₅). The complete set of data is given in Supporting Material.

Figure 2

Kinetic curves for apomyoglobin mutant with Leu¹¹⁵ replaced by Ala in the process of its folding (a) and unfolding (b) at various final urea concentrations (indicated by numbers near the curves). The process was monitored at pH 6.2 by integral Trp fluorescence intensity. (Solid lines) Single-exponential approximations of the slow-phase folding and of unfolding kinetics. (Inset) Burst-phase amplitude A versus urea concentration M in the folding process and baselines A_U(M), A_I(M) for amplitudes of unfolded and intermediate states.

Figure 3

(Upper panels) Observed folding/unfolding rate constant (k_obs) versus the final urea concentration M for apomyoglobin mutants. All rate constants are measured in s⁻¹ and given in a logarithmic scale. The left part of each chevron corresponds to folding experiments (open symbols), the right part to unfolding experiments (solid symbols). (Shaded symbols) Folding rate constants k_IN(M) for the I→N transition; these are calculated from Eq. 2 using the corresponding k_obs(M), extrapolations of unfolding rates k_NI(M) = k_NU(M), and f_I(M) plots shown for each protein (lower panel). The k_NI(M) values were calculated from the folding data in the region where the f_I(M) > 0.1. (Dashed lines) Linear approximations of ln(k_NI(M)), and (dash-dot lines) linear approximations of ln(k_IN(M)). (Dark-shaded lines) Chevron plots calculated using an equation k_obs = k_NI + f_I × k_IN. (Solid lines, upper panels) Chevron plots for WT protein (see WT panel). (Shaded lines, lower panels) Two-state fitting of f_I versus urea concentration. (Solid lines, lower panels) The f_I plots for WT protein (see WT panel).

Figure 4

The sperm whale apomyoglobin fold (adapted from (48)). The studied residues are shown as spheres; their color intensity (see the scale below the figure) is proportional to the Φ_I value, i.e., to the extent of their side-chain involvement in the intermediate state I (according to both kinetic and equilibrium data). The outer circles are proportional to the Φ_TS(UN) value, the extent of involvement of side chains of these residues in the folding nucleus in the U→N transition; the inner circles are proportional to the Φ_TS(IN) value, the extent of involvement of side chains in the folding nucleus in the I→N transition. Labels of conserved nonfunctional residues of A, G, H helices are underlined.