Cytochrome c Folding and Unfolding: A Biphasic Mechanism

How an unstructured polypeptide chain condenses to form a biologically functional protein, the protein folding problem, has attracted great interest in recent years.1 In many proteins, this spontaneous self-organization reaction occurs with high efficiency and fidelity despite a marginal thermodynamic driving force.2 A full knowledge of the free energy landscape associated with all possible configurations of the polypeptide backbone along the folding coordinate is required for a thorough understanding of this process.3,4 Unfortunately, calculations and measurements of the energy and entropy of dynamic structures on the molecular level are nontrivial tasks. It is, therefore, beneficial to construct general principles of folding based on simple model systems, such as cytochrome c. Cytochrome c (cyt c) is a small single-domain protein with a well-defined three-dimensional structure5 as illustrated in Figure 1. The protoporphyrin IX prosthetic group is covalently linked to the protein moiety through thioether linkages via Cys14 and Cys17. The robust protein architecture is made possible by the sophisticated H-bonding network throughout the molecule and the intramolecular linking between His18 and Met80 mediated by the heme central iron atom.6 Cyt c is an ideal model system for folding studies because it can be unfolded and refolded reversibly without degradation of the protein.7 Furthermore, the heme prosthetic group stays intact as the protein unfolds due to the covalent linkages between the heme and the protein moiety such that the refolding kinetics are not complicated by bimolecular rebinding of the heme to the polypeptide chain. Another advantage for using cyt c as a model system is that the various folding intermediates present during the course of folding can be regulated by a variety of chemical means, such as the pH and the concentration of denaturant.8-10 This provides the basis for understanding the contributions of the various onand off-pathway intermediates in the folding energy landscape.3,4 There is no universal probe that can provide snapshots of the conformation of each individual polypeptide chain with molecular resolution during the course of folding. However, a great deal of valuable information on specific aspects of cyt c folding has been obtained from a variety of spectroscopic techniques.11 For example, nuclear magnetic resonance (NMR) has been demonstrated to be very useful for probing solvent exposure of exchangeable protons on the polypeptide backbone.12 Techniques such as circular dichroism (CD)13 and small-angle X-ray scattering14 provide information on the secondary structure content and average molecular size of the protein molecule, respectively. Tryptophan fluorescence and optical absorption spectroscopies are informative concerning local interchain interactions by taking advantage of the presence of the heme prosthetic group.15,16 To open a new window, we sought to employ resonance Raman scattering, a structurally rich technique, to go beyond the phenomenological observations and examine quantitatively the microscopic details of the folding reaction.8-10,17,18 To study protein folding dynamics, the folding reaction must be initiated in a pulsed manner, such that all of the