Amyloid fibril depositions are associated with many neurodegenerative diseases. The nucleation step of fibrillation remains poorly characterized as no experimental technique allows for direct monitoring of nucleus formation. A new method based on 2D-correlation deep UV resonance Raman spectroscopy was applied to probe directly all species at early stages of lysozyme fibrillation, establish the sequential order of highly correlated events, and characterize quantitatively the kinetics of nucleus formation.
A new deep-UV Raman spectrometer utilizing a laser source tunable between 193 and 205 nm has been designed, built, and characterized. Only selected wavelengths from this range have previously been accessible, by Raman shifting of the second, third, and fourth harmonics of the Nd:YAG fundamental in hydrogen. The apparatus was demonstrated to be a useful tool for characterizing hen egg white lysozyme structural rearrangements at various stages of fibril formation. High-quality deep-UV resonance Raman spectra were obtained for both a protein solution and a highly-scattering gelatinous phase formed by fibrillogenic species. In addition to amide bands, strong contribution of nu(12) and ring-C phenylalanine vibrational modes was observed at excitation wavelengths below 200 nm. Remarkably, the Raman cross-section of these modes revealed dramatic change of lysozyme in response to heat denaturation and fibril formation. These results indicate that phenylalanine could serve as a new deep-UV Raman probe of protein structure.
A de novo, genetically engineered 687 residue polypeptide expressed in E. coli has been found to form highly rectilinear, beta-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy, and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage. The ordered self-assembly of designed, extremely repetitive, high molecular weight peptides is a harbinger of the utility of similar materials in nanoscience and engineering applications.
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