High resolution spectroscopy reveals fibrillation inhibition pathways of insulin

Abstract

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.

PIP2 Phospholipid‐Induced Aggregation of Tau Filaments Probed by Tip‐Enhanced Raman Spectroscopy

“…[20] The geometry and the electromagnetic properties of this nanoparticle defines its TERS activity,which may change from one TERS probe to another. This dependence,i na ddition to the sensitivity of TERS signals to molecular orientation and gradient-field effects,e xplains the plethora of TERS signatures with variable intensities reported for protein (amyloid especially) fibers described in the literature, [18,[21][22][23][24][25] and for K18 + POPC:PIP 2 and K18 + HS fibers in this study.Because of this intrinsic intensity variability of TERS bands,areliable comparison between TERS features identifiable in the two samples often requires the statistical investigation of their relative abundance in the spectra. Fort his purpose,w e collected 108 TERS spectra for the K18 + POPC:PIP 2 sample and 138 for its K18 + HS counterpart in distinct areas,u sing 25 TERS probes.…”

“…The proportion of TERS spectra with Raman bands at this wavenumber is higher in K18 + HS (59 %) than in K18 + POPC:PIP 2 (47 %) fibers,w hich bears out our conclusion regarding the difference between these two samples;t he information is,h owever,l argely blurred here by the contribution of other vibration modes,s uch as the CH 2 and CH 3 bending vibrations of amino acid side chains that are likely to appear in this spectral region. [18,22,33] Thei nvestigation of other vibration modes is discussed in the Supporting Information.…”

Amyloid formation kinetics of hen egg white lysozyme under heat and acidic conditions revealed by Raman spectroscopy

“…Many studies have successfully observed the morphological changes of lysozyme during amyloid fibrillation under heat and acidic conditions . On the basis of measurements of the hydrodynamic radius and total scattering intensity, Jain and Udgaonkar found a time delay between the formation of worm‐like protofibrils and their lateral association, providing conclusive evidence of a multistep mechanism of amyloid fibrillation.…”

“…Many studies have successfully observed the morphological changes of lysozyme during amyloid fibrillation under heat and acidic conditions. [21][22][23][24][25] On the basis of measurements of the hydrodynamic radius and total scattering intensity, Jain and Udgaonkar [26] found a time delay between the formation of worm-like protofibrils and their lateral association, providing conclusive evidence of a multistep mechanism of amyloid fibrillation. Dynamic light scattering and atomic force microscopy (AFM) experiments further suggested that the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation routine, in a so-called on-pathway from the amyloid monomers, oligomers, protofibrils, and more complex structures.…”

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

“…Deckert and co-workers investigated by TERS individual insulin fibrils, retrieving their amino acid residue composition. [31] The authors evidenced the conformational variety along the fibril by mapping alternating hydrophobic and hydrophilic domains and providing valuable information on the sites active for biological interactions. Recently, Bonhommeau et al showed via TERS the differentiation between the wild type of Aβ fibrils and two synthetic mutants, the latter obtained by substitution of a specific amino acid in the polypeptide sequence and notably forming toxic oligomers and nontoxic fibrils.…”