The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism

Abstract

Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of several neurological disorders. Many studies have assessed amyloid fibril formation under specific solution conditions, but they all lack an important phenomena in biological solutions—buffer specific effects. We have focused on the formation of hen egg-white lysozyme (HEWL) fibrils in aqueous solutions of different buffers in both acidic and basic pH range. By means of UV-Vis spectroscopy, fluorescence measurements and CD spectroscopy, we have managed to show that fibrillization of HEWL is affected by buffer identity (glycine, TRIS, phosphate, KCl-HCl, cacodylate, HEPES, acetate), solution pH, sample incubation (agitated vs. static) and added excipients (NaCl and PEG). HEWL only forms amyloid fibrils at pH = 2.0 under agitated conditions in glycine and KCl-HCl buffers of high enough ionic strength. Phosphate buffer on the other hand stabilizes the HEWL molecules. Similar stabilization effect was achieved by addition of PEG12000 molecules to the solution.

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation

“…[8,11,33,38] As recently reported by Brudar et al, buffer specific effects can dramatically affect amyloid self-assembly by stabilizing or disrupting the protein native structure, so that comparison of our results to previously described sequences is difficult. [39] Our fluorescence data are in accordance to conclusions of theoretical studies and show a correlation between hydrophobicity and amyloid formation rate. [9a,32] Figure 3e) at a concentration of 3 mM was found, which we explain based on its pronounced hydrophobicity, which is the strongest amount of all here studied phenylalanine derivatives (Figure 4a-b).…”

“…]: 10 mM ammonium acetate, pH 7.0) [8,11,33,38] . As recently reported by Brudar et al., buffer specific effects can dramatically affect amyloid self‐assembly by stabilizing or disrupting the protein native structure, so that comparison of our results to previously described sequences is difficult [39] …”

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

“…The fibrils of this protein also present a high pH dependence with respect to stability [ 155 ]. Proteins such as the Hen-egg lysozyme [ 156 ], the regulatory ATPase variant A (RavA) from E.coli [ 157 ], TTR [ 158 ], serum amyloid A [ 159 ], insulin [ 160 ], and glucagon [ 161 ], among others, have been shown to aggregate at acidic conditions (pH = 2–5), where the transition to a β-sheet structure upon oligomerization and amyloid aggregation was observed. However, other peptides and proteins, such as the designed EASZ model peptide [ 162 ], Ac-Phe-Phe-Cys-NH 2 (Ac-FFC-NH 2 ) [ 61 ] amyloid peptide model, and some peptides from human Pbx-regulating protein-1, have been shown to aggregate to β-sheet structure at basic pHs [ 163 ].…”

Buffer Influence on the Amino Acid Silica Interaction

“…[8] The latter comprises not only the peptide and the surface but also the solvent, which often contains a buffer to stabilize the pH of the system. The fact that buffer ions can compete with the peptide/ protein in binding to the surface is well known and investigated, [9][10][11][12][13][14][15][16][17][18][19][20] but to the best of our knowledge this has never been done for single amino acids especially for silica. A rational understanding of peptide or protein interactions with surfaces would benefit greatly from data on the interactions of individual amino acids.…”