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Kinetic paremeters of motochondrial porin incorporation into phospholipid membranes with different surface charge
No full textThe kinetics of pore formation by mitochondrial porin in lipid bilayer membranes with different polar heads was investigated. Pore formation was estimated by the total current flowing through the membrane. The time course of the current revealed a "pore activation process" or "cooperative process" and was well described by a novel mathematical model in which two of the four parameters, K1 and K2, are directly related to the processes of pore insertion into and disappearance from the membrane, respectively.
At 1M of KCl, for fixed porin concentration, the K1 value decreases in the following order: phosphatidylserine, phosphatidylcholine membrane, indicating a different affinity of protein to membranes bearing a different polar head.
According to the Guy-Chapman theory, at 0.1 M of KCI, the surface charge of negatively-charged membranes is -137 mV, as compared to -80 mV at 1 M of KCI, and the corresponding K1 value decreased considerably under these experimental conditions; on the other hand, in zwitterionic phospholipid membranes of phosphatidylcholine, the value of K1 remains unmodified.
When diluting the negative charge of phosphatidylserine by the addition of phosphatidylethanolamine (1: 1 w: v), the K1 value increased strongly. The K2 parameter seems to be less influenced by the magnitude of surface potential present on negatively-charged membranes or by the nature of the lipid head.
These results indicate that the electrostatic coupling between porin and the polar head of lipid membranes could play an important role in porin incorporation
Different states of assembly of mitochondrial porin in various lipid bilayer membranes
No full textIn natural membranes the large variety of proteins and lipids is correlated with different membrane functions. Although there is evidence that specific non-bilayer-forming lipids are needed for the correct functioning of certain cell proteins, it is not known how lipid properties influence protein functions. Several recent experiments have shown that lipid variations induce changes in the configuration of intrinsic membrane proteins.
In this work, we describe various different assemblies of mitochondrial porin into black lipid membranes. Bilayers, composed of a single type of lipid (phosphatidylinositol in n-decane or oxidized cholesterol in octane-n-decane), are formed in a medium of variable KCl concentration. In phosphatidylinositol membranes, the kinetics of porin incorporation up to the steady state showed sigmoidal curves, indicating cooperative phenomena. The steepness of the curves decreased with a reduction in the ionic strength of the membrane medium. From the best fit to data point with a four- parameter logistic equation, we obtained slope factors or Hill coefficients indicating the formation of different protein assemblies. In oxidized cholesterol membranes, mitochondrial porin seems to be present as a monomer. This configuration was found at both high and low ionic strengths. Although there is much evidence that the aggregation of protein is related to salt concentration (Lin S. et al., Anal Chim. Acta, 383:101-107,1999), given that solubility increases at low salt concentrations (>0.5M), and B-sheet proteins in particular predominantly increase their stability at salt concentrations of >1.5 M (Bandyopadhyay A.K. and Sonawat H.M., Biophys. J., 790.501- 510,2000), our results could indicate a coupling of the protein and lipid polar head
Mitochondrial porin incorporation in different black lipid membranes and its gating mechanism studied by means of alternating current
No full textThe kinetics of pore formation by mitochondrial porin in black lipid membranes of phosphatidylinositol and oxidized cholesterol was investigated as a function of external applied voltage. During the insertion process, the total current passing through the membrane is easily monitored by means of an ac device, and is interpreted as a probe of the process involved during channel incorporation and assembly. The time course of the total current flowing through the membrane was well-described by means of a mathematical model, that gives kinetic parameters describing two concurrent processes which can be interpreted as positive/negative cooperativity.
In the growth rate of pore incorporation we observe a process similar to a phase transition taking place at a critical time, an aspect which has been neglected in most previous investigations of insertion into model membranes.
The behavior around the instant of transition phase is compatible with what is typically expected in other kinetics growth processes. By using data obtained under steady state conditions, with a well-defined number of pores inserted into the membranes, the conductance measurements provided indirect indications of two possible gating mechanisms.
Moreover, our measurement device makes it easy to acquire information on the capacitance characteristics of black membranes. While before porin incorporation we do not observe a significant capacitance dependence on the external applied voltage, afterwards we find evidence of a dual-exponential voltage dependence of capacitance, a result similar to that found for conductance
Evidence for a different assembly of Gramicidin A in oxidized cholesterol black lipid membrane
No full textGramicidin 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
Kinetic of Gramicidin incorporation into black lipid membranes of oxidized cholesterol evaluated by means of alternating current
No full textInactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by pyridoxal 5'-phopsphate.
No full textMitochondrial porin incorporation into black lipid membranes: ionic and gating contribution to the total current
No full textWe present a new ac device useful for simultaneous measurements of ionic charge movement (conductance) and gating charge displacement (capacitance) in mitochondrial porin channels incorporated in two kinds of black lipid membranes (BLMs), made up of phosphatidylinositol (charged surface) and oxidized cholesterol (neutral surface). In particular, we investigated the conductance/capacitance variations during the process of porin incorporation (VDAC) at different porin concentrations. While conductance variations are present throughout the porin concentration range investigated, a threshold value seems to be necessary in order to detect a significant capacitance variation. A clear steady state in both conductance and capacitance is reached for the phosphatidylinositol bilayer, while for the oxidized cholesterol membranes, the steady state is reached only for the conductance. The dependence of capacitance characteristics on the membrane applied voltage V(m) is investigated before porin incorporation and at the ionic current steady state. The results obtained confirm that before porin incorporation, there is a small dependence on V(m)(2), while afterwards we find evidence of a dual exponential voltage dependence (a result similar to that found for conductance). Finally, we investigated the capacitance dependence on the radius of the hole separating the two compartments of the cell used in the measurements. In this study, performed only with oxidized cholesterol, the radius was varied from 200 to 1050 microm. We observed a significant variation in the specific capacitance in particular for smaller radii. The results were interpreted by a simple geometrical model taking into account the influence of the torus
Evidence for a different assembly of Gramicidin A in oxidized cholesterol black lipid membrane
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