Insights into the molecular mechanism behind solubilization of amyloidogenic polyglutamine‐containing peptides

Abstract

Synthetic peptides are widely used to understand the protein misfolding and aggregation observed in a diverse group of diseases called systemic amyloidosis and neurodegenerative disorders. The preparation of monomeric solutions of the aggregation‐prone peptides is necessary for achieving a mechanistic understanding efficiently, without compromising any critical step. This study describes novel approaches and mechanisms behind the conversion of otherwise insoluble/sparingly soluble peptides to soluble monomers by trifluoroacetic acid and hexafluoroisopropanol. Although the pretreatment with these solvents results in more or less similar monomers, the mechanism followed to achieve this differs with the solvent. Data show that these solvents aid in the solubility by disrupting the extensive intramolecular and/or intermolecular H‐bond network present in the insoluble peptides.

Nucleation-dependent aggregation kinetics of YeastSup35 fragment GNNQQNY

“…PGQ 9 I and PGQ 9 A peptides were solubilised using established protocols. [22][23][24][25][26] Mass spectrometry 5 µL of GNNQQNY peptide solubilized in water-HCl (pH 2.0) was injected into time-offlight (TOF) mass analyzer provided with electrospray ionization (ESI) source (Waters Q-TOF premier HAB213). The m/z spectrum recorded at a mass range of 50-3500 Da was plotted using OriginPro 8.5 data analysis and graphing software and the purity was estimated by analyzing the observed molecular ionization states of the peptide.…”

“…The absorbance of the peptide was recorded at 215 nm under a constant pressure of about 80 psi. 24,25…”

“…27 Light scattering (LS) and Thioflavin T (ThT) assays These assays were performed as described previously by taking 120 µL of ongoing aggregation reaction sample in a Quartz SUPRASIL Ultra-micro cell. 25 The intensity of light scattered by the sample was determined by setting the emission and excitation wavelengths at 450 nm and their slit widths at 2.5 nm. ThT fluorescence intensity was measured by adding ThT stock to a final concentration of 100 µM to the above sample.…”

“…3,33 Fourier-transform infrared (FTIR) spectroscopy FTIR spectroscopy analysis was carried out using Bio-ATR II cell mounted on Tensor 27, Bruker FTIR instrument. 22,25,34 GNNQQNY aggregates were subjected to washing thrice by using milliQ-water and then 50 µL was loaded on to the Bio-ATR II cell. A total of 120 scans at a resolution of 4 cm -1 under constant purging of nitrogen gas were collected at room temperature and averaged for the final FTIR spectra.…”

“…Samples for electron microscopy analysis were prepared by inverting the carbon-coated side of TEM grid on 5 µL of aggregate sample, incubated for thirty seconds and the excess sample was carefully blotted by using filter paper. 22,25,35 It was rinsed with 10 µL of milliQwater and then negatively stained by inverting on 5 µL of 2% uranyl acetate for 5-10 seconds. The stained grids were left overnight to air-dry and then imaged using a FEI-Tecnai G2 12 Twin (120 KV) transmission electron microscope.…”