The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure

Abstract

An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted hydrogen/deuterium exchange analyzed in dimethyl sulfoxide. Unlike its well studied homolog apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench-flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse-labeling experiments show that the slow-folding phase is associated with residues in the CE loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.

Figure 1. Backbone representations of Leghemoglobin and Myoglobin

The Lb structure was derived from the soybean nicotinate structure (PDB id 1FSL) and the Mb structure is the CO complex (PDB id 1MBC).

Figure 2. Interrupted H/D exchange studies for the native state of apoLb at pH 6.0

Data points are color coded according to the published helical structure of the holoprotein, as indicated by the colored bars at the top of the figure. Black data points are for turn and loop residues. Horizontal dotted lines are placed at protection factors of 6, 30 and 1000.

Figure 3. Kinetic Burst Phase Intermediate of apoLb Folding

(a) Colored points (left axis) show A0 values (proton occupancy in the kinetic burst phase of folding, corrected for the effects of high-pH pulse duration22) for apoLb, plotted against residue number. The continuous black line shows the per-residue average area buried upon folding (AABUF), calculated as a continuous running average of 9 residues. Horizontal lines are plotted at A0 values of 0.4 and 0.7. Where A0 > 0.7 (red points), the amide proton is considered to be completely protected within the dead time of the quench flow apparatus (6.4 ms) (termed fast-folding or F). Where A0< 0.4, the amide proton is considered to be protected only in the slow, or visible, phase of folding (termed slow folding or S). Green points (0.4 < A0 < 0.7) indicate intermediate behavior, where some members of the ensemble are protected early and some at a later time (termed F+S). The locations of helices in the folded holoprotein are indicated with black bars at the top of the graph. (b) Backbone representation of soybean Lb, with amide proton protection (F, F+S, S) plotted according to the colored points of Figure 3a. Helices are labeled; arrows designate the direction N→C. Red spheres indicate the three amides in the center of the E helix that are most highly protected from exchange in the burst phase intermediate.

Figure 4. Equilibrium Intermediate (pH 3.7) from Interrupted H/D Exchange

(a) Colored points show protection factors for amide proton exchange as a function of residue number: blue, PF < 6; green, 6 < PF < 30; red, PF > 30. Horizontal dotted lines are placed at protection factors of 6 and 30. (b) Backbone representation of soybean Lb, with protection factors plotted according to the colored points of Figure 4a. Helices are labeled; arrows designate the direction N→C.

Figure 5. Comparison of Equilibrium and Kinetic intermediates of apoLb

For each segment of the polypeptide chain, the amide proton protection factors (for the equilibrium intermediate; Figure 4) and the A0 values (for the kinetic intermediate, Figure 3) are plotted onto the backbone structure, color coded according to the corresponding values in Figures 3 and 4. Each segment is labeled at its N-terminus.

Figure 6. Time Course of apoLb Folding

(a) Transitions of proton occupancy for the designated regions of the apoLb amino acid sequence, as a function of the folding time t_fold. Curves were fitted using published methods only to those data sets with proton occupancy less than 0.8 at 6.4 ms. The majority of curves (shown with thinner lines) have rate constant k ~ 2–3 s⁻¹. Measurably slower folding rates (k < 1 s⁻¹) are designated with thicker lines. Curves labeled with two residue numbers are derived from overlapped cross peaks in the HSQC spectra. (b) Plot of folding rate constant k as a function of residue. Black bars show well-determined rates for amides where the proton occupancy at t_fold = 6.4 ms is less than 0.7. Green bars show less well-determined rates for amides with proton occupancy at t_fold = 6.4 ms between 0.7 and 0.8. Blue bars show well-determined rates less than 1 s⁻¹ (corresponding to thicker curve-fits in Figure 6a). Red dots show amides with proton occupancy at t_fold = 6.4 ms > 0.8, for which no folding rates were calculated. The positions of helices in the folded holoprotein are indicated at the top of the graph, and loops are labeled.

Figure 7

(a) Stopped flow-pH-jump time course for soybean apoLb, normalized to an amplitude of 1.0. (b, c) residuals for fitting with (b) a single exponential and (c) two exponential functions. The burst phase amplitude is 0.6, the single exponential amplitude is 0.4, with a rate constant of 1.11 s⁻¹. For the double exponential fit, the faster phase is fit with amplitude 0.25 and a rate constant of 2.04 s⁻¹, and the slower phase with an amplitude of 0.15 and rate constant of 0.64 s⁻¹.

Figure 8. Location of the slowest-folding amides on the structure of soybean Lb

The position of the amide nitrogen is shown, with the spheres colored according to the helix colors of Figure 1.