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|Title:||Interactions of class A and class L amphipathic helical peptides with model membranes|
|Authors:||Polozov, Ivan V.|
|Advisor:||Epand, Richard M|
|Abstract:||<p>Amphipathic α-helix, that is an α-helix with opposing hydrophilic and hydrophobic faces oriented along the long axis of the helix, is an often encountered secondary structural motif in biologically active peptides and protein. In this thesis, there were systematically studied membrane interactions of two amphipathic α-helical peptides: the 18L peptide, which belongs to the class L (lytic peptides), and the Ac-18A-NH₂ peptide of the class A (apolipoprotein) according to classification of Segrest et al. (1990). It was found previously [Tytler et al., 1993] that class A and class L peptides have opposing effects on some properties of biological and model membranes. In order to elucidate the molecular mechanism responsible for this so-called reciprocal effect in biological membranes, we studied a number of aspects of membrane interactions of 18L and Ac-18A-NH₂ peptides including equilibrium and kinetics membrane binding, permeabiIization, fusion and conductance. These functionally different peptides also displayed a number of similarities in membrane interactions and thus parallel studies of these two peptides provide an insight into the general features of amphipathic peptide membrane interactions. It was found that membrane binding equilibrium and kinetics for both peptides can be described as monomer partitioning with saturation at high peptide/lipid ratios. The rate of peptide-membrane association is relatively close to the diffusion limit Increase in membrane affinity correlates with a decrease in dissociation rate, i.e. with slower peptide exchange. Both for cationic 18L and zwitterionic Ac-18A-NH₂, the presence of acidic lipids increased membrane binding constants by two orders of magnitude. We have shown that the dynamic character of the peptide membrane equilibrium can be used for selective peptide targeting and disruption of membranes with specific lipid composition. Titration calorimetry experiments show that the free energy of binding results from an entropic contribution. The free energy of peptide-membrane association is in the range of 8. 5-12. 8 kcal/mol. In regards to effects on membrane domain organization, 18L and Ac-18A-NH₂ peptides displayed more similarities than differences. While binding with high affinity to fluid membranes, peptides were unable to penetrate into the lipid membrane in the gel state. If trapped kinetically by cooling from the fluid phase, peptides dissociated from the gel membrane on the time scale of several hours. Charge-charge interaction were capable of inducing lateral domain formation in fluid membranes. Both peptides had affinity for anonic lipids which resulted in about 30 percent enrichment of acidic lipids within several nanometers of the peptide's tryptophan, but there was no long-range order in peptide-induced lipid demixing. Peptide insertion in fluid acidic membranes was accompanied by only a small increase in bilayer surface and a decrease in polarity in the membrane core. Peptide-lipid charge-charge interactions were also capable of modulating existing domain composition in the course of the main phase transition in mixtures of anionic phosphatidylglycerol with zwitterionic phosphatidylcholine. We were unable to observe any peptide-induced lateral phase separation in fluid zwitterionic membranes with nonbilayer phase propensity. Peptides actually improved lipid mixing within the temperature range of the main phase transition of the DMPC:DMPE binary system. Peptide-membrane permeabilizing activity was significantly affected by the presence of acidic lipids and, in zwitterionic membranes, the presence of nonbilayer forming lipids. In anionic membranes (DOPC:DOPG, DOPG) both peptides caused leakage in the range of bound peptide/lipid ratios of 1: 100 to 1: 1 0 and on the time scale of seconds. In zwitterionic vesicles in the range of bound peptide/lipid ratios 1 :50 to 1: 10, 18L caused both leakage and fusion on the time scale of hundreds of seconds. At the same peptide/lipid ratios. higher lipid concentrations favoured more fusion and less leakage. Ac-18A-NH₂ caused vesicle leakage, but not fusion. The lytic activity of 18L increased and that of Ac-18A-NH₂ decreased with an increase in the content of an inverted phase forming lipid. A reciprocal effect of 18L and Ac-18A-NH₂ was observed in both the vesicle leakage and vesicle fusion assays with zwitterionic lipids. Reciprocal effects of 18L and Ac-18A-NH₂, were restricted only to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE,DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests that nonbilayer phase propensity is the physical property responsible for observation of reciprocal effects of A- and L-class peptides in biological membranes. Analysis of the mechanism of 18L induced permeabilization of zwitterionic membranes have shown that 18L peptide destabilises membranes. leading to a transient formation of large defects (diameter> 3 om) which generally results in contents leakage. but in the presence of bilayer-bilayer contact can lead to vesicle fusion. This defect formation is accompanied by phospholipid flip-flop and or peptide translocation. Peptide insertion into the membrane also significantly reduces membrane stability to mechanical tension making it susceptible to osmotic lysis. This mechanism may be general for the action of class L peptides.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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