The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.
Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.
Mature human apolipoprotein C‐I (apoC‐I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC‐I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC‐I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC‐I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC‐I with yields of approximately 4–5 mg per L of culture. The purified product has properties similar to plasma‐derived apoC‐I including self‐association in the lipid‐free state and induced α‐helical content in the presence of egg‐yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short‐chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro‐PCho), to examine the interaction of apoC‐I with submicellar phospholipid. Circular dichroism spectroscopy and cross‐linking experiments show that apoC‐I acquires helical content and remains self‐associated at submicellar concentrations of Hex2Gro‐PCho (4 mm). Sedimentation equilibrium studies of apoC‐I at submicellar levels of Hex2Gro‐PCho and analysis of the effects of apoC‐I on the 1H NMR spectrum of Hex2Gro‐PCho indicate micelle induction by apoC‐I, and establish the capacity of apoC‐I to assemble individual phospholipid molecules.
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