Fiber diffraction as a screen for amyloid inhibitors.

Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimer's, Parkinson's, Huntington's, etc., as the early-stage, oligomers that form before the development of beta-conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.7 A corresponding to the hydrogen-bonding spacing between the beta-chains, and the other at approximately 10 A corresponding to the spacing between beta-pleated sheets. We report here the application of fiber x-ray diffraction to monitor these structural indicators of amyloid fiber assembly in the presence of small, aromatic molecules, some of which have been assessed by other techniques as being inhibitory. The compounds included butylated hydroxytoluene, chloramphenicol, cotinine, curcumin, diphenylalanine (FF), ethyl 3-aminobenzoate methane sulfonate, hexachlorophene, melatonin, methylpyrrolidine, morin, nicotine, phenolphthalaine, PTI-00703 (Cat's claw), pyridine, quinine, sulfadiazine, tannic acid, tetracaine, tetrachlorosalicylanilide, and tetracycline. Their effects on the aggregation of Abeta1-40, Abeta11-25, Abeta12-28, Abeta17-28, Abeta16-22, and Abeta16-22[methylated] analogues were characterized in terms of the integral widths and integrated intensities of the two characteristic reflections. Peptide Abeta11-25 with or without small molecules showed varying relative intensities but similar coherent lengths of 28-49 A in the intersheet and 171-221 A in the H-bonding directions. PTI-00703, however, abolished the H-bonding reflection. Among previously reported aromatic inhibitors for Abeta11-25, PTI-00703, tannic acid, and quinine were more effective than curcumin, morin, and melatonin based on the criterion of crystallite volume. For the N-methylated and control samples, there were no substantial differences in spacings and coherent lengths; however, the relative volumes of the beta-crystallites, which were calculated from the magnitude of the intensities, decreased with increase in concentration of Abeta16-22Me. This may be accounted for by the binding of Abeta16-22Me to the monomer or preamyloid oligomer of Abeta16-22. The fiber diffraction approach, which can help to specify whether an amyloidophilic compound acts by impeding hydrogen-bonding or by altering intersheet interactions, may help provide a rationale basis for the development of other therapeutic reagents.

Cytoplasmic domain of zebrafish myelin protein zero: adhesive role depends on beta-conformation.

Solution spectroscopy studies on the cytoplasmic domain of human myelin protein zero (P0) (hP0-cyt) suggest that H-bonding between beta-strands from apposed molecules is likely responsible for the tight cytoplasmic apposition in compact myelin. As a follow-up to these findings, in the current study we used circular dichroism and x-ray diffraction to analyze the same type of model membranes previously used for hP0-cyt to investigate the molecular mechanism underlying the zebrafish cytoplasmic apposition. This space is significantly narrower in teleosts compared with that in higher vertebrates, and can be accounted for in part by the much shorter cytoplasmic domain in the zebrafish protein (zP0-cyt). Circular dichroism measurements on zP0-cyt showed similar structural characteristics to those of hP0-cyt, i.e., the protein underwent a beta-->alpha structural transition at lipid/protein (L/P) molar ratios >50, and adopted a beta-conformation at lower L/P molar ratios. X-ray diffraction was carried out on lipid vesicle solutions with zP0-cyt before and after dehydration to study the effect of protein on membrane lipid packing. Solution diffraction revealed the electron-density profile of a single membrane bilayer. Diffraction patterns of dried samples suggested a multilamellar structure with the beta-folded P0-cyt located at the intermembrane space. Our findings support the idea that the adhesive role of P0 at the cytoplasmic apposition in compact myelin depends on the cytoplasmic domain of P0 being in the beta-conformation.