Multiple pathways on a protein-folding energy landscape: kinetic evidence

Abstract

The funnel landscape model predicts that protein folding proceeds through multiple kinetic pathways. Experimental evidence is presented for more than one such pathway in the folding dynamics of a globular protein, cytochrome c. After photodissociation of CO from the partially denatured ferrous protein, fast time-resolved CD spectroscopy shows a submillisecond folding process that is complete in approximately 10(-6) s, concomitant with heme binding of a methionine residue. Kinetic modeling of time-resolved magnetic circular dichroism data further provides strong evidence that a 50-microseconds heme-histidine binding process proceeds in parallel with the faster pathway, implying that Met and His binding occur in different conformational ensembles of the protein, i.e., along respective ultrafast (microseconds) and fast (milliseconds) folding pathways. This kinetic heterogeneity appears to be intrinsic to the diffusional nature of early folding dynamics on the energy landscape, as opposed to the late-time heterogeneity associated with nonnative heme ligation and proline isomers in cytochrome c.

Figure 1

(A) TRMCD spectra of cytochrome c at logarithmic time intervals (330 ns to 25 ms, eight times/decade) after CO photolysis (arrows show change with time). Global kinetic analysis finds spectral changes (b-spectra) corresponding to four exponential time constants: (B) 3 μs, (C) 50 μs, (D) 300 μs, and (E) 700 μs (dark lines). The first b-spectrum closely resembles a (normalized) [five-coordinate heme − Met–His] model difference spectrum (B, light line), having a strong minimum near 415 nm that arises from the 413-nm lobe of the positive MCD A term of the native protein (37), consistent with the formation of native-like coordination. The second b-spectrum lacks this feature, resembling a [five-coordinate − bis-His] difference spectrum (C, light line) based on model spectra from cytochromes c₃ (44) and b₅ (38), and is assigned to the formation of nonnative histidine coordination. The final two CO pressure-dependent rate processes are dominantly CO recombinations (D and E).

Figure 2

Intermediate MCD spectra calculated from the heterogeneous kinetic mechanism in Fig. 3: (A) The aggregate five-coordinate species, cyt_Met* + cyt_His* + cyt_U*, (B) cyt[Met–Fe–His], (C) cyt[His–Fe–His]*, and (D) cyt*-CO (A–D, dark lines). These were optimized to fit the spectra of corresponding model cytochromes (A–D, light lines); see text. Intermediate spectra calculated from a multiple-equilibria mechanism (Scheme S1) are shown for comparison (A–D, - - -) (These overlay the spectra from the heterogeneous mechanism in all panels except C.).

Figure 3

Heterogeneous kinetic model in which multiple folding pathways after photodissociation of partially denatured cytochrome c-CO (cyt*-CO) are distinguished by geminate positioning of different residues (Met, His) for facile binding to heme iron. Thermal equilibration between configurational ensembles cyt_Met* and cyt_His* takes place before photolysis but is slow compared with heme binding processes after photolysis. Folding of cyt_Met* ensembles is fast, whereas folding of cyt_His* ensembles is slow compared with CO recombination at 1 atm (not shown). A small fraction of unfolded configurations, cyt_U*, lacks geminate positioning of a labile residue and remains unligated at the sixth axial heme site (until CO recombines).

Scheme 1

Homogeneous multiple-equilibria model for heme ligation events after photolysis of cytochrome c-CO.

Figure 4

Schematic diagram of the internal free energy (E) landscape for folding after cytochrome c-CO photolysis, cyt*-CO _→^hν cyt* + CO, as a function of protein configurational coordinates (CC). A folding funnel (N) and an overlapping local minimum (T), corresponding to the histidine-ligation trap, together form a surface topologically similar to the “moat” landscape of Dill and Chan (6) describing heterogeneous (fast and slow) folding kinetics.