Mechanisms of m-cresol-induced Protein Aggregation Studied Using a Model Protein Cytochrome c

Abstract

Multi-dose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol is one such AP, but has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. m-cresol induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of m-cresol decreased the temperature at which the protein aggregated and increased the aggregation rate. However, m-cresol did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an “invisible” partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation “hot-spot”. Based on these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation.

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

“…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). The extent of aggregation seems to qualitatively correlate with the hydrophobicity of the AP (Singh et al 2011, Hutchings et al 2013). APs also have been suggested to interact with hydrophilic regions of proteins (Alford et al 2011).…”

“…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).…”

Insights into the molecular mechanism of protein native-like aggregation upon glycation

“…In the present work we have focused our attention on cytochrome c, a protein recently used as a model to study protein aggregation (26). Model proteins offer several advantages as they are abundant and inexpensive, and their biophysics have been well characterized.…”

Influence of freezing down to 77.15 K on structure and antioxidant power of some proteins

“…Other parameter for Cyt C conformation investigation is an absorption band at 695 nm which measures the stability of a local protein region in Cyt C. This absorption band corresponds to the charge transfer from the side chain sulfur of the methionine residue (Met-80) to the heme ferric iron. Molecular loosening in Met-80 region leads to the absorption band absence due to lack of ligation of the amino acid to the heme [18]. Cyt C freezing leads to decreasing of absorbance band intensity (Fig.…”