CHARACTERIZATION OF A PARTIALLY UNFOLDED STRUCTURE OF CYTOCHROME-C INDUCED BY SODIUM DODECYL-SULFATE AND THE KINETICS OF ITS REFOLDING

The mechanism of unfolding of ferricytochrome c induced by the surfact ant sodium dodecyl sulfate has been studied by heme absorption, trypto phan fluorescence, circular dichroism, resonance Raman scattering, sto pped-flow and time-resolved resonance energy transfer to obtain a comp rehensive view of the whole process. Unfolding occurred at an almost s pecific molecular ratio of SDS/cytochrome c in the concentration range (20-50 mu M) studied here. However there appears to be a point at app roximate to 0.6 mM SDS where unfolding begins to occur for lower cytoc hrome c concentrations. The kinetics of unfolding revealed only a sing le transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximate to 16 kJ/mol, indicat ing that other associated steps, if any, are too fast to be significan tly populated. The free energy change (Delta G degrees) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25 %). indicating that a significant amount of helical struc ture remains folded in contrast to a complete loss of helical structur e in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spe ctra, shows a major population (approximate to 95 %) of mis-ligated hi stidine to the heme which acts as a kinetic trap in the folding proces s. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in t he native protein to 0.63 ns in the unfolded one, associated with a mo vement of Trp59 by 10 Angstrom away from heme. The maximum entropy met hod analysis of fluorescence decay indicated the growth of various con formational substates in SDS-unfolded cytochrome c in contrast to narr owly distributed conformations in the native protein. The refolding wa s comprised of three kinetic steps; the first was significantly fast ( approximate to 8 ms) and was assigned to the dissociation of His26 tha t paves the protein towards correct folding pathway. The other two slo wer steps probably arise from chain misorganization and prolyl isomeri zation. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytoc hrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.