Probe amphipathic

CTC, used extensively to monitor calcium release in both whole cells and isolated organelles (28-33), is an amphipathic molecule that easily passes through cell membranes (see Figure 1). The fluorescence of this probe is enhanced more than fiftyfold by binding of calcium when the dye is intercalated into biological membranes. 

The development of amphipathic fluorescent dyes that label endocytic vesicles has permitted the study of endo-cytosis in nerve terminals in real time [25,26], The probe FM1-43 equilibrates between the aqueous phase and the membrane but is not membrane-permeating. The plasmalemma becomes fluorescent (Fig. 10-8). Upon endocytosis, the labeled membrane is internalized. When removed from the extracellular medium, the dye is retained by the endocytic vesicles but lost from the plasmalemma. Endocytic vesicles are transformed into synaptic vesicles containing FM1-43. Importantly, recycled synaptic vesicles lose the probe upon exocytosis. 

Figure 1. (Bottom) Diagram of the electrostatic potential adjacent to a membrane bearing a positive charge. The zeta potential is the potential at the hydrodynamic plane of shear, which should be about 2 A from the surface of the membrane. (Top) Schematic of the location of the probe molecules used to detect the potential produced by the adsorption of calcium and other alkaline earth cations to membranes formed from PC. The divalent cation cobalt and the amphipathic, anionic, fluorescent probe TNS will sense the potential at the interface. The non-actin-Rf complex will sense the potential in the center of the membrane.

Melittin, which is an amphipathic peptide from honeybee venom, consists of 26 amino acid residues and adopts different conformations from a random coil, to an a-helix, and to a self-assembled tetramer under certain aqueous environments see Fig. 9. We have carried out our systematic studies of the hydration dynamics in these three conformations using a single intrinsic tryptophan ( W19) as a molecular probe. The folded a-helix melittin was formed with lipid interactions to mimic physiological membrane-bound conditions. The self-assembled tetramer was prepared under high-salt concentration (NaCl = 2 M). The tryptophan emission of three structures under three different aqueous environments is 348.5 nm, 341 nm, and 333.5 nm, which represents different exposures of aqueous solution from complete in random-coil, to locating at the lipid surface of a nanochannel (50 A in diameter) in a-helix and to partially buried in tetramer. Figure 10 shows... 

The emission of Trp 19 in melittin shifts to the red side peaking at 341 nm (Fig. 18), and the probe location slightly moves away from the lipid interface toward the channel center. Consistently, we observed a larger fraction of the ultrafast solvation component (35%) and a smaller contribution of slow ordered-water motion (38%). Melittin consists of 26 amino acid residues (Fig. 9), and the first 20 residues are predominantly hydrophobic, whereas the other 6 near the carboxyl terminus are hydrophilic under physiological conditions. This amphipathic property makes melittin easily bound to membranes, and extensive studies from both experiments [156-161] and MD simulations [162-166] have shown the formation of an 7-helix at the lipid interface. Self-assembly of 7-helical melittin monomers is believed to be important in its lytic activity of membranes [167-169]. Our observed hydration dynamics are consistent with previous studies, which support the view that melittin forms an 7-helix and inserts into the lipid bilayers and leaves the hydrophilic C-terminus protruding into the water channel. The orientational relaxation shows a completely restricted motion of Trp 19, and the anisotropy is constant in 1.5 ns (Fig. 20b), which is consistent with Trp 19 located close to the interface around the headgroups and rigid well-ordered water molecules. 

A major effect of cholesterol on the conformation of apoE was revealed by comparing the conformation on DMPC discs, on HDLc, and on spherical artificial microemulsion particles by circular dichroism (Mims et ai, 1990). Conformational differences of apoE on different types of particles also were demonstrated using NMR to probe lysyl microenvironments. When the apoE lysyl residues were labeled by reductive methylation with [ C]formaldehyde to allow detection, the lysyl microenvironments manifested dramatic differences on a discoidal particle compared to spherical particles (S. Lund-Katz et aL, 1993). On spherical particles, two lysine microenvironments were observed, but on discoidal particles eight peaks were observed (apoE has 12 lysyl residues). These results indicate that apoE structure differs significantly on the two lipid surfaces. In a systematic study of the effect of the particle lipid composition on the conformation of apoE, conformation was shown to be affected by a number of parameters (Mims et ai, 1990). The a-helical content was lower when apoE was bound to a spherical particle compared to a discoidal particle. It was concluded that this probably reflects the different ways in which the amphipathic helices interact with phospholipid on the two particles. With discoidal particles the interaction is primarily with phospholipid acyl side chains, whereas with spherical particles the interaction is with polar phospholipid head groups. In addition, the conformation of apoE was influenced by the diameter of the microemulsion particle and possibly by the order/ disorder of the lipid components. 

Although both melittin and the cecropins share a structural motif that consists of two helices linked by a hinge region, the polarity of the helices is reversed in that melittin has a hydrophobic N-terminus and an amphipathic C-terminus the converse is true for the cecropins. The relationship of structural difference to the different activities of these two peptides was probed by preparation of shortened cecropin-melittin hybrids (200, 201) that show enhanced antibacterial activity and decreased hemolytic effects. 

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