The effect of the osmolyte trimethylamine N‐oxide on the stability of the prion protein at low pH

Abstract

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.

The Effects of Trimethylamine N-Oxide on the Structural Stability of Prion Protein

“…Significantly, addition of 1.5 M and 2.0 M weakens the entire α-helical conformation. The unfolding of α-helical structure has been reported with similar work performed at low pH (Granata et al 2006). Consistently, the significant unfolding of prion proteins is carried out at > 1.0 M TMAO as illustrated in Figure 4B.…”

“…This study suggests a potential stratagy for preventing PrP Sc by stabilizing the conformation of PrP C . In contrast, later studies indicated that TMAO destabilized the α-helical conformation of fulllength human prion at neutral pH at high temperature (Nandi et al 2006) and human prion proteins at low pH (Granata et al 2006). The structure of α-helix-rich prion protein is destabilized and the prion protein is converted to β-sheet structured oligomeric species (Nandi et al 2006).…”

Molecular Dynamics as an Approach to Study Prion Protein Misfolding and the Effect of Pathogenic Mutations

“…Both simulations were carried out with protonation states of the amino acids corresponding to the strongly acidic pH regime, to perturb the PrP structure. TMAO itself was not protonated (whereas its pK a is ~4.7), as this is likely to cause destabilization ra-ther than stabilization of protein structure [169,170]. Starting from the misfolded conformation, the extended sheet is disrupted and PrP regains contacts that had been lost during the pH-induced misfolding (Fig.…”

Manipulation of lysozyme phase behavior by additives as function of conformational stability

“…This pH dependent osmolyte action has been related to the chemical nature of the osmolytes, e.g. the pK a values (Granata et al, 2006;Natalello et al, 2009;Singh et al, 2009), but could not explain all pH dependent observations (Kaushik and Bhat, 2003). In contrast, there are indications that the pH dependent mode of action of additives might have its origin in the nature of the proteins instead (Kaushik and Bhat, 2003;Macchi et al, 2012).…”

“…So far, pH dependent osmolyte action was connected to the chemical nature of the osmolyte, e.g. the pK a value (Granata et al, 2006;Natalello et al, 2009;Singh et al, 2009). This could be excluded in our case as no pK a is exceeded between pH 3 and pH 5 for the investigated additives.…”