Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

Abstract

Bovine β‐lactoglobulin (BLG) is a major whey protein with unique structural characteristics: it possesses a free Cys thiol (SH) and two disulfide (SS) bonds and consists of a β‐barrel core surrounded by one long and several short α helices. Although SS‐intact conformational folding has been studied in depth, the oxidative folding pathways and accompanying SS formation/rearrangement are poorly understood. In this study, we used trans‐3,4‐dihydroxyselenolane oxide, a water‐soluble selenoxide reagent which undergoes rapid and quantitative SS formation, to determine the oxidative folding pathways of BLG variant A (BLGA) at pH 8.0 and 25 °C. This was done by characterizing two key one‐SS intermediates, a particular folding intermediate having a Cys66–Cys160 SS bond (I‐1) and a particular folding intermediate having a Cys106–Cys119 SS bond (I‐2), which have a native Cys66–Cys160 and Cys106–Cys119 SS bond, respectively. In the major folding pathway, the reduced protein (R) with abundant α helices was oxidized to I‐1, which was then transformed to I‐2 through SS rearrangement. The native protein (N) was formed by oxidation of I‐2. The redundant Cys121 thiol facilitates SS rearrangement. N is also generated from an ensemble of folding intermediates having two SS bonds (2SS) intermediates with scrambled SS bonds through SS rearrangement, but this minor pathway is deteriorative due to aggregation or overoxidation of 2SS. During oxidative folding of BLGA, α→β conformational transition occurred as previously observed in SS‐intact folding. These findings are informative not only for elucidating oxidative folding pathways of other members of the β‐lactoglobulin family, but also for understanding the roles of a redundant Cys thiol in the oxidative folding process of a protein with odd Cys residues.

The Non-native Disulfide-Bond-Containing Landscape Orthogonal to the Oxidative Protein-Folding Trajectory: A Necessary Evil?

“…Today, researchers in the field of oxidative folding continue to push the envelope by developing novel redox reagents that mimic traditional redox reagents and catalysts such as protein disulfide isomerase (PDI). , Selenium-based redox reagents have been particularly popular. − The use of novel redox reagents and manipulation of the regenerative conditions have unraveled rich information about the trajectories adopted by disulfide-bond-containing proteins, including the dependence of the trajectory on such factors. − These results give rise to the notion that, in the endoplasmic reticulum, regeneration might take place through heretofore unknown pathways. The application of denaturing agents or mutation of amino acids in the WT protein has often caused the regeneration rate to increase.…”

Improving the Annotation of the Venom Gland Transcriptome of Pamphobeteus verdolaga, Prospecting Novel Bioactive Peptides

“…Furthermore, odd cysteine numbers have also been described previously in cones [57]. Even when not usually described in toxins, unpaired cysteines play a role in other processes such as the polymerization of immunoglobulins [58], the stabilization of oxidized proteins [59], and in signal transduction associated to the sensing of reactive oxygen species [60], making them not completely uncommon.…”

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments

“…Therefore, the oxidative folding pathways of a protein with odd Cys residues were not well known until recently. Probably the first extensive study of such a case was reported using bovine milk β-lactoglobulin (BLG) as a model protein [ 72 ].…”

“…In addition to this major folding pathway, N could also be regenerated from the scrambled 2SS intermediate ensemble via SS rearrangement, but this minor pathway would be deteriorative because the 2SS was prone to self-aggregate irreversibly probably due to the presence of a free Cys SH group. During the oxidative folding of BLGA, the redundant Cys121 SH group may assist efficient SS-rearrangement of the SS intermediates, guiding them to I-1 and then I-2, not to 2SS [ 72 ].…”