A protein’s natively folded state, as we call it, may or may not be an energy-minimized state. It can very well be a state with higher energy than some intermediates, giving the protein intrinsic dynamic flexibility or induced dynamic response to stimuli. Thus, the quest to find out 1) cellular mechanisms that render the native state under kinetic control and 2) the factors responsible for stabilizing the native state under biological timescales, despite the existence of alternative stable states, is still ongoing.

Disulfide bonds, covalently linking the two cysteine side chains, contribute significantly to protein folding by reducing conformational entropy. Depending on when disulfide bond formation occurs during the folding, they may be considered one of the factors that lay a kinetic trap for the folding. For example, in certain folding reactions, spontaneous formation of correct disulfides upon providing reduced buffer conditions may guide the protein to a native state, because the cysteines are suitably positioned a priori. In other reactions, kinetic intermediates may promote temporarily incorrect or random disulfide formation, which gets resolved at a later stage in the folding, either unassisted or with the help of chaperones. There is one more category of proteins in which native disulfides cannot be formed at all, even after providing oxidizing buffer conditions in vitro. Such proteins are stabilized into alternative conformations of which the possibility of forming a disulfide bond is nil. These proteins must rely on cellular mechanisms to ensure that correct disulfides are formed before folding. One such cellular mechanism is the co-translational formation of disulfides with the help of certain enzymes while the proteins are being secreted out of ribosomes in the endoplasmic reticulum (ER). Although many studies have shown that the formation of disulfides at this stage significantly enhances folding efficiency, specific molecular details of such processes are missing in the literature.

In the article titled “Co-translational formation of disulfides guides folding of the SARS-CoV-2 receptor binding domain,” Shakhnovich and co-workers iron out the molecular details of the coupling of disulfide formation and the folding of the receptor binding domain (RBD) of the SARS-CoV-2. Using fluorescence assays, the authors show that the RBD can be refolded reversibly from a denatured state only if its native disulfide bonds are present before folding. The authors further show that oxidative refolding from a denatured and fully reduced state leads to misfolding into a non-native molten-globule-like state, which forms fewer disulfide bonds than the native state. Thus, the study suggests that the native state might be kinetically trapped by its disulfide bonds. Furthermore, simulations predicted that the native cysteine pairs more frequently come in close proximity, suggesting that co-translational oxidation in the ER may help the RBD arrive at its metastable native state. Thus, the study advances our understanding of SARS-CoV-2 pathology and provides a detailed molecular picture of disulfide-coupled protein folding.