Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One

Abstract

In practice and despite Anfinsen’s dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.

Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze–Thaw Method

“…This suggested the possible presence of soluble aggregates and/or misfolded and/or unstable protein species (Fink, 1998;Toichi et al, 2013;Saotome et al, 2019). It was hypothesized that such refolded species could be due to the presence of reducing agent like 1 mM DTT in the freeze-thaw buffer, high protein concentration, or longer refolding time duration.…”

Bioactivity Characterization of Purified Recombinant Hypothetical Protein Coded by Open Reading Frame-112 of Streptomyces.

“…For instance (4) suggested that, the maximum SDS-binding concentrations are generally estimated at 1.4 g SDS / g protein. It is known that disulfide bonds decrease SDS binding to proteins by up to 2-fold (22). The pI of proteins affects mobility too, because the presence of many charged AAs leads to disruption of their binding to SDS micelles (15).…”

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins

“…In particular, our study aims to examine whether a protein (any protein) becomes toxic upon aggregation/ destabilization. We chose DENV3-ED3 as a model protein for a practical reason: Several variants with single or few mutations that exhibited uniquely different biophysical properties in terms of oligomerization and thermal stability [ 15 , 16 , 17 ] were available, and DENV3-ED3′s native structure was well characterized [ 18 ]. Altogether, this study provided insights into the mechanisms of cellular and in particular mitochondrial damage caused by non-amyloid-misfolded proteins.…”