A structural model for apolipoprotein C-II amyloid fibrils: experimental characterization and molecular dynamics simulations.

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-ß-structure composed of two parallel ß-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-ß-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' ß-strand-loop-ß-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-ß-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-ß-scaffold containing relatively unstructured connecting loops.

Solid-state NMR and simulation studies of equinatoxin II N-terminus interaction with lipid bilayers.

The interaction with model membranes of a peptide, EqtII(1-32), corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [(19)F-para]Phe16-[1-(13)C]Leu19 and [(19)F-para]Phe16-[(15)N]Leu23 analogs of EqtII(1-32) measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16-23, in the presence of bilayers. (19)F-(31)P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII(1-32) in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII(1-32) in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC-bound peptide adopting helical formations at residues 16-24, whereas the DMPC:SM-bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Membrane protein structure determination using solid-state NMR.

Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr > approx 30,000) complex unless specific methods to reduce the information content to a manageable amount are employed. Such methods include specific residue-type labeling, labeling of putative segments of a protein, or examination of complexes made up of smaller, manageable units, such as oligomeric ion channels. Labeling possibilities are usually limited to recombinant or synthesized proteins, and labeling strategies often follow models from a bioinformatics approach. In all cases, and in common with most membrane studies, sample preparation is vital, and this activity alone can take considerable effort before NMR can be applied--peptide or protein production (synthesis or expression) followed by reconstitution into bilayers and resolution of suitable sample geometry is still technically challenging. As experience is gained in the field, this development time should decrease. Here, the practical aspects of the use of solid-state NMR for membrane protein structural determinations are presented, as well as how the methodology can be applied. Some successes to date are discussed, with an indication of how the area might develop.

Neurotoxic, redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation.

The amyloid beta peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Abeta peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Abeta) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Abeta. However, unlike the unoxidized peptide, Met(O)Abeta is unable to penetrate lipid membranes to form ion channel-like structures, and beta-sheet formation is inhibited, phenomena that are central to some theories for Abeta toxicity. Our results show that, like the unoxidized peptide, Met(O)Abeta will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Abeta production contributes to the elevation of soluble Abeta seen in the brain in Alzheimer's disease.

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin.

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes.

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.

A solid-state NMR study of protein mobility in lyophilized protein-sugar powders.

The molecular mobility of protein in lyophilized lysozyme-sugar systems stored at different relative humidities was studied using solid-state NMR. Relaxation measurements, T(1) of high-frequency (MHz), and T(1rho), of low-frequency (kHz) motions, were performed on lysozyme lyophilized with lactose and trehalose. Molecular aggregation and enzymatic activity of the protein were determined using HPLC and bioassays. An increase in hydration had little effect on the T(1rho) values of pure lysozyme, trehalose, lactose, trehalose-lysozyme, and lysozyme at low lactose concentrations. The T(1) values of pure sugar increased as moisture content increased. The presence of both sugars led to increased T(1) values of the lysozyme but increasing hydration gradually reduced T(1) values. When a larger amount of lactose was lyophilized with lysozyme, longer T(1) (and T(1rho)) values were seen for lactose than for lysozyme. Although longer T(1) values were related to an increase in protein stability, the effect of crystallization and sugar type appeared to be major contributing factors. Trehalose and lactose decreased relaxation rates in the lysozyme-sugar systems while hydration increased relaxation rates that were correlated with changes in aggregation and activity of the protein.