The interactions of horse heart apocytochromec with phospholipid vesicles and surfactant micelles: time-resolved fluorescence study of the single tryptophan residue (Trp-59)

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Abstract
The interactions of horse heart apocytochromec with membrane interfaces were studied on membrane models including micelles of the anionic surfactant sodium dodecyl sulfate (SDS), the micelle forming lipid analogs dodecylphosphoglycol (Cl2PG), tetradecylphosphoglycol (Cl4PG), and dodecylphosphocholine (Cl2PN), and the negatively charged phospholipid 1-palmitoyl-2-oleoylsn-glycero phosphocholine (POPS) forming small unilamellar vesicles (SUV). The time-resolved fluorescence of the single tryptophan residue (Trp-59) emission was monitored to characterize the modifications of the conformational equilibrium and of the internal dynamics of the protein, which can be brought about by its binding to these model membranes. In most of the cases, as for the protein in solution, the excited state lifetime distribution of the Trp emission was described by four discrete classes, whose relative proportions and barycenters vary significantly in the different complexes formed. In the complex with POPS, however, the decay analysis showed only 3 lifetime classes: the long lifetime class displayed a bary-center value smaller than that observed for the protein in aqueous solution but with a much higher proportion, indicating a stabilization of this conformer in the membrane-bound form of the protein. A similar sensitivity of theTrp-59 excited state to deactivation by thermal collisions in water and in the protein/POPS complex was observed, indicating a probable location ofTrp-59 at the membrane/water interface. The effects of protein binding to C12PN, C12PG and C14PG micelles on the long life-time class proportion were similar to that of POPS but, in addition, there was a large contribution of a short lifetime component which was absent in POPS vesicles. The bary-center values of the excited state lifetime classes were comparable in these membrane systems, suggesting thatTrp-59 is not transferred to a non-polar environment. Binding of apocytochrome c to SDS micelles strongly reduced the lifetime class barycenters and, in contrast to the other membrane systems, strongly favored the contribution of the shortest lifetime class at the expense of the c3 class. This suggests an interaction of theTrp-59 with the sulfur containing head-group of this surfactant. The indole ring mobility is reduced at the interface contacts. A fastTrp-59 mobility with a large amplitude is suggested in the complex with POPS by an initial anisotropy value lower than the expected one of 0.295 measured in vitrified medium. These observations can be correlated with the induction of α-helical structure after interactions of apocytochromec with membrane model systems (de Jongh and de Kruijff 1990).

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