Raman Spectroscopic Studies of Amyloids

National Heart, Lung, And Blood Institute

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Abstract

Raman microspectroscopy is a powerful technique that couples the chemical specificity of Raman spectroscopy with the spatial resolution of a microscope. Raman spectroscopy is commonly used to measure protein amide bands, 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. In the past reviewing period, we reported an approach using uniformly 13C2H15N-labeled alpha-synuclein. The advantage here is the capability of resolving alpha-synuclein from endogenous proteins in a cellular environment due to the presence of the 13C-2H stretching bands, which appear in the center of the cellular quiet spectral region, where no naturally-occurring biomolecules are vibrationally active. This isotopically labeled protein was used to examine fibril endocytosis, which is thought to be a key component of disease progression in synucleinopathies. Preformed 13C2H15N-labeled fibrils were fed to cultured mammalian cells and mapped by Raman spectral imaging to examine the fate of these endocytosed fibrils. Our data demonstrate that the 13C2H15N-amide-I band can be resolved from other cellular vibrational signals and that the strong 13C2H stretching bands enable background-free mapping of exogenous -syn in cells. The internalized fibrils largely accumulate at the cellular periphery, colocalizing with proteins and lipids. This work demonstrates unambiguous localization of internalized fibrils and direct observation of beta-sheet structure of amyloid fibrils in cells. Because this strategy of incorporating bio-orthogonal vibrational probes into proteins is broadly applicable, we will continue to refine and optimize this methodology in order to gain insights into the mechanisms of protein misfolding and amyloid formation as they relate to disease.