Role of partial protein unfolding in alcohol‐induced protein aggregation

Abstract

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as non-disease-related proteins. Here we probed the biophysical mechanisms underlying alcoholinduced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.

Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

“…In a Bacillus subtilis strain, the lethal concentration of BzA increased the membrane fluidity (43). For protein structures, BzA induced the aggregation of proteins, such as human interleukin (44) and interleukin-1 receptor antagonist (45), by partially unfolding these proteins (46,47). Unfolding actions were also reported for short-chain alcohols (48).…”

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy

“…However, the presence of transient oligomer formation during the NMR experiments resulting in a decrease in NMR peak volumes cannot be completely ruled out. Increased protein dynamics 30,37–40 in the presence of polysorbates populating an ensemble of conformations with the average conformation close to that in the absence of polysorbates can also lead to decreased crosspeak volumes. In addition, the pattern of the volume decrease is not uniform across all the crosspeaks, indicating varying protein dynamics in various regions of the protein.…”

Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein

“…However, it has been shown that these preservatives cause protein aggregation (Maa and Hsu 1996, Katakam and Banga 1997, Remmele Jr. et al 1998, Gupta and Kaisheva 2003, Tobler et al 2004, Roy et al 2006, Thirumangalathu et al 2006). We have demonstrated previously that APs used in liquid protein formulations lead to protein destabilization and aggregation using the model protein Cyt c (Singh et al 2010, Singh et al 2011, Hutchings et al 2013). The extent of this effect was dependent upon the nature of the AP, and the pattern of aggregation observed was CR > PH > BA > PE.…”

“…APs do not have strong binding sites on proteins (Maa and Hsu 1996, Roy et al 2006, Singh et al 2010, Singh et al 2011). APs may hydrogen bond with the peptide backbone, suggested by earlier studies on BA and polyproline monitoring changes in amide I, amide II, and hydroxyl bands using infrared spectroscopy (Strassmair et al 1969).…”