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.
Highlights
In the present contribution, we fibrillized insulin under standardized conditions[18] in the presence of benzonitrile (PhCN), β-carotene or quercetin (Que), and dimethylsulfoxide (DMSO)
Gel development indicated fibril growth, and the sample was characterized with AFM, TERS and standard Raman spectroscopy in the dry state to obtain information on the morphology and the core and surface conformations, respectively
The AFM measurements of the sample obtained from the PhCN-insulin fibrillation approach (Fig. 3) reveal that the small molecule PhCN slowed the progression of insulin fibril formation but did not entirely prevent it
Summary
We fibrillized insulin under standardized conditions[18] in the presence of benzonitrile (PhCN), β-carotene or quercetin (Que), and dimethylsulfoxide (DMSO). The formed species were thoroughly characterized using AFM, TERS and conventional Raman spectroscopy. If β-carotene or Que in DMSO was present, unfolding of the native insulin was initiated; the process stopped yielding unordered stable aggregates (i), (ii). Either β-sheet rich stable insulin aggregates were generated or the fibrillation was completely suppressed. DMSO did not affect the mature fibrils under the chosen conditions
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