Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.
Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme.
Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.
Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥ 40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.
Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535-540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT-CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.
The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiencywas measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.
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