Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering

Abstract

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.

Figure 1

A schematic of the diffusive mixer.

Figure 2

Time-resolved Kratky plots. (a) A Kratky plot of the protein in the inlet channel, to the left of the cross in Fig. 1, is shown, indicating that the initial (pH 2) state is expanded and random coil-like. (b) The SAXS pattern after the device had been translated to illuminate the protein stream as indicated in Fig. 1, 200 μm from the center of the cross. This x-ray exposure averaged over a time interval from 150 to 500 μs, assuming that the protein stream traveled at its mass averaged velocity. Roughly 90% of the volume of the protein solution was above pH 3 in this interval. We have subtracted 10% of the initial pH 2 state from the observed Kratky plot at this position to generate the curve shown in b. A simple calculation based on these average numbers and using the rate constants for a pH jump to 4.5 (15, 18), indicates that 45% of the protein above pH 3 at this location was in the denatured state and 55% was in the native state. The measured R_g from the data of b (R_g^app = 15.9 ± 0.7 Å) is related to that of the denatured (R_g^D) and native (R_g^N) states as follows: (R_g^app)² = f_D(R_g^D)² + f_N(R_g^N)², where f_D/N represents the fraction of the sample in the denatured/native state (22). Our calculation of f_D is a lower limit (hence our calculated R_g^D = 18.1 ± 0.9 Å is an upper limit) for the following reasons: (i) We have ignored the contribution from the expanded denatured state in calculating the R_g of the compact denatured state from R_g^app. (ii) We expect the folding to occur more slowly with a final pH of 3 than for the calculated final pH of 4.5. (iii) Imidazole may not completely bind before non-native histidines produce misfolded structures that fold more slowly than the imidazole complex (–15). (iv) The calculation assumes that all of the protein is mixed at t = 0, the position at which the thinnest, 2.5-μm section is mixed. The mixing is actually a continuous process, with thicker sections mixing at later times. (c) The Kratky plot at a position that corresponds to a time of ≈10 msec after mixing (red, noisy curve). This data suggests that the protein had folded completely to the native state. A Kratky plot of a much larger sample of native cytochrome c in equilibrium at pH 7 is shown as the green (smooth) curve plotted on top of the data. The best fit to the data in b and c are shown as dashed, blue lines.