Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip‐Enhanced Raman Spectroscopy

vandenAkker, Corianne C.; Deckert‐Gaudig, Tanja; Schleeger, Michael; Velikov, Krassimir P.; Deckert, Volker; Bonn, Mischa; Koenderink, Gijsje H.

doi:10.1002/smll.201500562

Cited by 72 publications

(92 citation statements)

References 65 publications

(102 reference statements)

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“…In contrast to ROA and CD, TERS exclusively probes the surface of a sample enabling a differentiation of core and surface composition of proteins, as has been demonstrated for amyloid fibrils. 17,18 …”

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Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

et al. 2016

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The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.

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Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

et al. 2016

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“…Another common feature of amyloid fibrils is a highly organized hydrogen bonded β-sheet structure core, which is detectable in the bulk sample with various techniques, including solid-state nuclear magnetic resonance (NMR)2, X-ray diffraction2, vibrational circular dichroism12, infrared spectroscopy13, and Raman spectroscopy1415. Single-particle studies on the nanoscale with tip-enhanced Raman scattering (TERS) on insulin fibrils161718 and human islet amyloid precursor peptide (hIAPP) fibrils19 revealed that the surface is not only composed of β-sheet structures but also has a high propensity for α-helix/unordered structures. Whereas conventional Raman spectroscopy is highly specific but limited to bulk samples, the intrinsic high sensitivity of TERS not only provides access to single amyloid fibrils but also allows the distinction between amino acids and conformations on fibril surfaces.…”

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High resolution spectroscopy reveals fibrillation inhibition pathways of insulin

Deckert‐Gaudig

Deckert

2016

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Fibril formation implies the conversion of a protein’s native secondary structure and is associated with several neurodegenerative diseases. A better understanding of fibrillation inhibition and fibril dissection requires nanoscale molecular characterization of amyloid structures involved. Tip-enhanced Raman scattering (TERS) has already been used to chemically analyze amyloid fibrils on a sub-protein unit basis. Here, TERS in combination with atomic force microscopy (AFM), and conventional Raman spectroscopy characterizes insulin assemblies generated during inhibition and dissection experiments in the presence of benzonitrile, dimethylsulfoxide, quercetin, and β-carotene. The AFM topography indicates formation of filamentous or bead-like insulin self-assemblies. Information on the secondary structure of bulk samples and of single aggregates is obtained from standard Raman and TERS measurements. In particular the high spatial resolution of TERS reveals the surface conformations associated with the specific agents. The insulin aggregates formed under different inhibition and dissection conditions can show a similar morphology but differ in their β-sheet structure content. This suggests different aggregation pathways where the prevention of the β-sheet stacking of the peptide chains plays a major role. The presented approach is not limited to amyloid-related reasearch but can be readily applied to systems requiring extremely surface-sensitive characterization without the need of labels.

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“…Figure a,b reports a collection of selected TERS spectra acquired from type A and type B oligomer samples. In spite of the local signal variations that are consequential to the chemical complexity produced by adjacent amino acid residues, a tentative assignment of the main TERS bands was made based on the Raman spectra (see Table ). In particular, aromatic ring vibrations are visible at 1003 and 1027 cm −1 (Phe) and within the 1550–1620 cm −1 range (Trp, Tyr, Phe) .…”

Section: Resultsmentioning

confidence: 99%

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip‐Enhanced Raman Spectroscopy

D’Andrea

Foti

Cottat

et al. 2018

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Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.

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Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip‐Enhanced Raman Spectroscopy

Cited by 72 publications

References 65 publications

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

High resolution spectroscopy reveals fibrillation inhibition pathways of insulin

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip‐Enhanced Raman Spectroscopy

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