STRUCTURE AND STABILITY OF CYTOCHROME-C FOLDING INTERMEDIATES

Hydrogen exchange labeling and nuclear magnetic resonance (NMR) approaches were used to elucidate the folding mechanism of horse cytochrome c (cyt c). The development of hydrogen bonded structure during refolding was observed by pulse labeling at variable refolding times, and the degree of protection from NH exchange was probed by systematic variation of the labeling pH. The results show that the folding reaction involves both sequential and parallel pathways. About 50% of the molecules follow a sequential pathway where a partially folded intermediate is formed in a 20 ms folding phase. This intermediate has two native-like helices near the chain termini, but lacks stable H-bonded structure in other parts of the molecule. Amide sites on either helix are not only protected at the same rate, but they also exhibit the same degree of protection, confirming previous evidence that association of the N- and C-terminal helices is an important early event in cyt c folding. Subsequent folding events on the 100 ms time scale involve replacement of a non-native histidine heme ligand by the native methionine ligand. In addition, there is evidence for a minor species of very rapidly folding molecules that form native-like structure within the mixing dead-time, as well as slow-folding forms that take several seconds to fold.