Raman Spectroscopic Studies of Amyloids

National Heart, Lung, And Blood Institute

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Abstract

We have used Raman microspectroscopy to characterize amyloid formation of alpha-synuclein, which is implicated in Parkinsons disease. This direct spectroscopic method reports on intrinsic molecular vibrations such as protein amide bonds, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. Specifically, we have coupled the Raman spectrometer with an inverted microscope, which yields both chemical and spatial information within macroscopic amyloid aggregates. Despite its high chemical specificity, Raman spectroscopy on its own does not offer site-specific information that is afforded by fluorescence and nitroxide-spin probes. To overcome this limitation, we have used native chemical ligation to produce segmentally 13C-labeled alpha-synuclein to provide region-specific structural details. As the 13C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues during amyloid fibril formation. Interestingly, the formation of insoluble beta-sheet aggregates precedes filament formation (as observed by TEM) and enhanced thioflavin-T emission. Beta-sheet structure first forms in the N-terminal region, followed by the C-terminal region of ligated-alpha-synuclein. Further, we see that within a single, microscopic aggregate, the C-terminal region is less uniform than the N-terminal region. From this work, we gained both molecular and mechanistic insights into alpha-synuclein amyloid formation and we anticipate this strategy will be broadly applicable to other studies of amyloid formation.