Spectroscopic studies on the stability and nucleation-independent fibrillation of partially-unfolded proteins in crowded environment

Synergistic Effect of Charged Amino Acids on the Stability and Fibril Formation of Lysozyme

“…It is observed that lysozyme lost all the four disulfide bonds with the addition of 100‐fold excess of DTT (Figure S2). This DTT‐reduced unfolded form significantly lost its secondary structure and exposes its hydrophobic core as reported earlier [49,64] . This confirmation drives the protein to form fibrils rapidly via nucleation‐independent pathway through the interchain interactions of the exposed hydrophobic core [49,65] .…”

“…Lysozyme fibrils were obtained by incubating 140 μM of the protein at 50 °C with 100-fold in excess of DTT in 20 mM sodium phosphate buffer (pH 7) as described previously. [49] The fibril formation kinetics was monitored by following the increase in ThT fluorescence at 485 nm after exciting the dye at 440 nm. [54] The fibrillation reaction was also probed in the presence of varying concentrations of individual amino acids and mixtures of amino acids as shown in Table 1.…”

Imidazolium-based ionic liquids with increasing alkyl chain length of cations decrease the stability and fibrillation propensity of lysozyme

“…Both nucleation-dependent and nucleation-independent pathways of fibrillation have been reported for Lyz. 34,47 In our earlier study, we reported that Lyz could form fibrils in the absence of any denaturant at neutral pH when its disulfide bonds were reduced using DTT and incubated at 50 °C. 47 This was first verified by the increase in fluorescence of ThT, which specifically binds to amyloid-like fibrils (Fig.…”

“…S8, ESI †), as reported earlier. 47 This partially unfolded conformation has a higher b-sheet content than the native state, which might facilitate the formation of fibrils. The fibrillation rate of Lyz under these conditions is found to be faster compared with the rates obtained under various other conditions.…”

“…1B) were chosen for studying the fibril formation of Lyz reported under pH 7 conditions. 47 Fibril formation of the protein in aqueous solution containing various concentrations of the ILs was analyzed. Also, the stability and structural changes of the protein and the binding affinity with ILs were examined through spectroscopic and molecular-docking studies.…”