We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.

The stable tyrosine radical YD. (tyrosine 160 in the D2 polypeptide) in photosystem II (PSII) exhibits nonexponential electron spin-lattice relaxation transients at low temperature. As previously reported, the tetranuclear Mn complex in PSII significantly enhances the spin-lattice relaxation of YD.. However, in Mn-depleted PSII membranes, the spin-lattice relaxation transients of YD. are also nonexponential, and progressive power saturation (P 1/2) experiments show that it does not behave like an isolated tyrosine radical. A model is developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a random orientation in a magnetic field. This model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of three relaxation rate constants: the "intrinsic" relaxation rate, the relaxation rate due to scalar exchange, and the relaxation rate due to dipole-dipole interactions. The intrinsic and the scalar exchange relaxation rates are isotropic and together contribute a single rate constant to the spin-lattice relaxation transients. However, the dipolar relaxation rate is orientation dependent. Each orientation contributes a different dipolar relaxation rate constant to the net spin-lattice relaxation rate constant. The result is a superposition of single-exponential recoveries, each with a different net rate constant, causing the observed saturation-recovery transients to be non-(single)-exponential. Saturation-recovery relaxation transients of YD. are compared with those of a model tyrosine radical, generated by UV photolysis of L-tyrosine in a borate glass. From this comparison, we conclude that scalar exchange does not make a significant contribution to the spin-lattice relaxation of YD. in Mn-depleted PSII. We account for the nonexponential relaxation transients obtained from YD. in Mn-depleted PSII membranes in terms of dipolar-induced relaxation enhancement from the non-heme Fe(II). From simulations of the spin-lattice relaxation transients, we obtain the magnitude of the magnetic dipolar interaction between YD. and the non-heme Fe(II), which can be used to calculate the distance between them. Using data on the non-heme Fe(II) in the reaction center of Rhodobacter sphaeroides to model the non-heme Fe(II) in PSII, we calculate a YD.-Fe(II) distance of greater than or equal to 38 A in PSII. This agrees well with the distance predicted from the structure of the bacterial reaction center.

Diatoms are unicellular phytoplankton accounting for approximately 40% of global marine primary productivity [1], yet the molecular mechanisms underlying their ecological success are largely unexplored. We use a functional-genomics approach in the marine diatom Phaeodactylum tricornutum to characterize a novel protein belonging to the widely conserved YqeH subfamily [2] of GTP-binding proteins thought to play a role in ribosome biogenesis [3], sporulation [4], and nitric oxide (NO) generation [5]. Transgenic diatoms overexpressing this gene, designated PtNOA, displayed higher NO production, reduced growth, impaired photosynthetic efficiency, and a reduced ability to adhere to surfaces. A fused YFP-PtNOA protein was plastid localized, distinguishing it from a mitochondria-localized plant ortholog. PtNOA was upregulated in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), a molecule previously shown to regulate intercellular signaling, stress surveillance [6], and defense against grazers [7]. Overexpressing cell lines were hypersensitive to sublethal levels of this aldehyde, manifested by altered expression of superoxide dismutase and metacaspases, key components of stress and death pathways [8, 9]. NOA-like sequences were found in diverse oceanic regions, suggesting that a novel NO-based system operates in diatoms and may be widespread in phytoplankton, providing a biological context for NO in the upper ocean [10].

Magainins are cationic, membrane-active peptides which show broad-spectrum antimicrobial activity. We have investigated the secondary structure and location of an analogue of magainin 2 in synthetic phospholipid bilayers using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Ala19-magainin 2 amide exhibits both alpha-helix and beta-sheet secondary structures in lipid bilayers containing either dipalmitoylphosphatidylglycerol (DPPG) or a 1:1 molar mixture of DPPG and dipalmitoylphosphatidylcholine (DPPC). The combination of FTIR and solid-state NMR results suggests that there are two populations of peptide. The secondary structure of one population is alpha-helix while that of the other population is beta-sheet. We demonstrate that the solid-state NMR technique, rotational-echo double resonance (REDOR), can be used to measure both intra- and intermolecular dipole-dipole interactions in membrane-bound peptides. Our REDOR experiments indicate that alpha-helical Ala19-magainin 2 amide is bound near the phospholipid head groups.

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