Amyloid-beta-sheet formation at the air-water interface.

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)).

Selective staining by the fluorochrome, 5,5-diphenyl-9-ethyl-DiOC2(3). I. Physicochemical studies of dye-dye and dye-tissue interactions.

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC2(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein beta-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.

Selective staining by the fluorochrome 5,5-diphenyl-9-ethyl-oxacarbocyanine. II. Application to paraffin embedded nervous tissue.

We present a simple and rapid method to label specific structures of neural tissue in paraffin sections. After incubation of deparaffinized sections in the cyanine dye 5,5'-diphenyl-9-ethyl-oxacarbocyanine (DEOC), three different washing and mounting procedures were performed. Incubation in DEOC followed by washes in ethanol and water and mounting in glycerol-gelatin resulted in selective labeling of myelin in the central and peripheral nervous systems. Weak labeling of myelin and axons and staining of nuclei of neurons was seen after incubation in DEOC followed by washes in ethanol and xylene, and mounting in Eukitt. Nuclear proteins, the endoplasmic reticulum and the Golgi apparatus were stained strongly and exclusively after incubation in DEOC, washes in water, ethanol and xylene, and mounting in Eukitt. Thus the absorption pattern of DEOC is changed significantly by solvents applied after the fluorochrome. In any case, fluorescence did not fade even after repeated and intense fluorescence microscopy over a period of 6 months.

Examination of polypeptide beta-sheet structure in solutions and thin layers: determination of the concentration and the 'critical aggregation concentration' using a cyanine dye as sensor.

In the visible spectra of some cyanine dyes a bathochromic shift of the dye monomer band was observed on the preconditions that: (1) beta-sheet containing polypeptides (denotes also proteins) were presented; and (2) these polypeptides were embedded in a layer or aggregated in solution. The band with the polypeptides which contained only the alpha-helix did not shift. In several cases the absorbance lifetime of the shifted band was limited. This was caused by dye self-association at the polypeptide surface, but there were enough quantities for this lifetime to obtain exact analytical measurements. These were executed quantitatively (100-20% beta-sheet), qualitatively (to about 10% beta-sheet) and moreover for the determination of the 'critical aggregation concentration' (cac). The applications of the dye sensor in biophysics, medicine and pharmacy were discussed.

Band-shifting through polypeptide beta-sheet structures in the cyanine UV-Vis spectrum.

If oxa- or thiacarbocyanine is introduced into an aqueous poly-L-lysine (PL) solution in a concentration higher than that of aggregation, then a shift of the absorption band of the cyanine monomer (M) can be observed in the UV-Vis spectrum, provided that the PL has a beta-sheet conformation. Other polypeptide aggregates with a high beta-sheet content exhibit this effect as well, whereas for PL with an alpha-helix conformation no spectral shift is observed. The force-field optimized molecular models and the calculated interaction energies prove that the beta-sheet interacts significantly more intensively with the cyanine than the alpha-helix does. The quantum chemically calculated highest occupied and lowest unoccupied molecular orbital (HOMO-LUMO) energies of the cyanines and cyanine beta-sheet polypeptide complexes predict a M-shift to bathochromic frequencies in agreement with experimental findings. In the case of the measured M-shift to hypsochromic frequencies, the shift appears to be influenced by the presence of cyanine J-aggregates. The results open the way for a fast and simple method to identify polypeptide beta-sheet structures in biological and other systems containing polypeptides by using cyanine as a sensor.