Gramicidin A (GA) is a hydrophobic linear pentadocapeptide that forms cation- selective channels in natural and black lipid membranes (BLM). The channels are believed to consist of single-stranded helices with the NH2 terminals linked together by hydrogen bonds. Linear gramicidins require a lipid bilayer to fold properly. If the phospholipid acyl chains are less than eight carbons long, channel folding is not observed; if the acyl chains are too long, the membrane- spanning channels are destabilized. Besides, embedded proteins might influence the molecular packing lipids (R. E. Koeppe II et al. Biol. Skr. Dan. Selsk, 49-93-98, 1998). In our experiments, we chose oxidized cholesterol black lipid membranes because their thickness (40±10 A) (Ti Tien and A. L. Diana, In Chemistry and Physics of Lipids, 2, 1968) is suited to both gramicidin length (25-30 A) and folding. The time course of gramicidin incorporation into BLM, studied by means of alternating current (E. Gallucci et al., Biophys. J. 71:824- 831, 1986), was sigmoidal independently of the KCI concentration used, indicating a cooperative process of incorporation into BLM. By means of a simple mathematical model (S. Micelli et al, submitted) we estimated the tm value, i.e. the parameter that describes a phase transition process (or similar phenomenon). This phenomenon has been observed by AA as a function of temperature and GA concentration (J. A. Killian and B. De Kruijff, Biophys. 1. 53: 111-117, 1988; Mingtao Ge and J. H. Freed, Biophys. J., 76: 266-280, 1999). On the other hand, the average values of the steady-state conductance against gramicidin concentration, for different KCI concentrations, shows a sigmoidal behavior too. These curves were fitted by means of a four-parameter equation which allowed us to obtain the Hill coefficient, D, or slope factor. The Hill coefficient gives the number of single-stranded helices which form the channel. Our results suggest that the number of channel sub-units depends on the KCI concentration. In our case, at a KCI concentration of 1M, the channel is characterized by two sub-units, in accordance with AA's finding (D. U. Urry, Proc. Natl. Acad. Sci. USA, 86:672, 1971, W. R. Veatch and R. Blond, Biochemisty, 13:5249, 1974), while for KCI concentrations of 0.1 and 0.5 M. the channel is formed by more than two sub-units.
Evidence for a different assembly of Gramicidin A in oxidized cholesterol black lipid membrane
MELELEO D.;
1999-01-01
Abstract
Gramicidin A (GA) is a hydrophobic linear pentadocapeptide that forms cation- selective channels in natural and black lipid membranes (BLM). The channels are believed to consist of single-stranded helices with the NH2 terminals linked together by hydrogen bonds. Linear gramicidins require a lipid bilayer to fold properly. If the phospholipid acyl chains are less than eight carbons long, channel folding is not observed; if the acyl chains are too long, the membrane- spanning channels are destabilized. Besides, embedded proteins might influence the molecular packing lipids (R. E. Koeppe II et al. Biol. Skr. Dan. Selsk, 49-93-98, 1998). In our experiments, we chose oxidized cholesterol black lipid membranes because their thickness (40±10 A) (Ti Tien and A. L. Diana, In Chemistry and Physics of Lipids, 2, 1968) is suited to both gramicidin length (25-30 A) and folding. The time course of gramicidin incorporation into BLM, studied by means of alternating current (E. Gallucci et al., Biophys. J. 71:824- 831, 1986), was sigmoidal independently of the KCI concentration used, indicating a cooperative process of incorporation into BLM. By means of a simple mathematical model (S. Micelli et al, submitted) we estimated the tm value, i.e. the parameter that describes a phase transition process (or similar phenomenon). This phenomenon has been observed by AA as a function of temperature and GA concentration (J. A. Killian and B. De Kruijff, Biophys. 1. 53: 111-117, 1988; Mingtao Ge and J. H. Freed, Biophys. J., 76: 266-280, 1999). On the other hand, the average values of the steady-state conductance against gramicidin concentration, for different KCI concentrations, shows a sigmoidal behavior too. These curves were fitted by means of a four-parameter equation which allowed us to obtain the Hill coefficient, D, or slope factor. The Hill coefficient gives the number of single-stranded helices which form the channel. Our results suggest that the number of channel sub-units depends on the KCI concentration. In our case, at a KCI concentration of 1M, the channel is characterized by two sub-units, in accordance with AA's finding (D. U. Urry, Proc. Natl. Acad. Sci. USA, 86:672, 1971, W. R. Veatch and R. Blond, Biochemisty, 13:5249, 1974), while for KCI concentrations of 0.1 and 0.5 M. the channel is formed by more than two sub-units.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
