A peptide‐display protein scaffold to facilitate single molecule force studies of aggregation‐prone peptides

Abstract

Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinson's disease (PD), which is associated with the aggregation of a-synuclein (aSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein-protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation-prone region of aSyn (residues 71-82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length aSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of aSyn 71-82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein c-synuclein (cSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of cSyn 71-82 is lower than that of aSyn [71][72][73][74][75][76][77][78][79][80][81][82] , but that a mixed aSyn 71-82 : cSyn 71-82 dimer forms with a similar propensity to the aSyn 71-82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation-prone peptides, which would otherwise be difficult to study.

Investigating the interactions of the first 17 amino acid residues of Huntingtin with lipid vesicles using mass spectrometry and molecular dynamics

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Single-Molecular Heteroamyloidosis of Human Islet Amyloid Polypeptide

“…The aggregation of amyloid proteins can be directly observed or inferred from transmission electron microscopy (TEM), X-ray diffraction (XRD), circular dichroism spectroscopy, Fourier transform infrared spectroscopy, atomic force microscopy (AFM), − as well as nuclear magnetic resonance spectroscopy. The kinetics of amyloid protein aggregation, when probed by a thioflavin T (ThT) or Congo red fluorescence assay, follows a sigmoidal trajectory to indicate the three stages of (primary and secondary) nucleation, , elongation, and saturation, where disordered monomers aggregate to render α-helical and then β-sheet-rich oligomers, protofibrils, amyloid fibrils, and plaques.…”

Molecular-Scale Investigations Reveal the Effect of Natural Polyphenols on BAX/Bcl-2 Interactions

“…While the peak forces for kaempferol, quercetin, dihydromyricetin, baicalin, curcumin, rutin, EGCG, and gossypol were 66.60 ± 0.64, 61.87 ± 4.50, 61.92 ± 1.71, 54.86 ± 1.29, 57.28 ± 1.19, 47.66 ± 0.86, 53.06 ± 0.57, and 58.59 ± 4.86 pN, respectively. These data are in the same strength as the interactions between amyloidogenic proteins [ 30 ] or redox proteins [ 31 ] and lower than the enzyme–coenzyme complexes [ 32 ], which is to be expected. Obviously, the addition of polyphenols resulted in decreases of 6.66%, 13.29%, 13.22%, 23.11%, 19.72%, 33.20%, 25.72%, and 17.88%, respectively.…”