Characterizing the role of cysteines and disulfide bonds in the folding and assembly pathway of P22 tailspike

Non-native disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, a homotrimer of 210 kDa, non-native disulfide bonds form both in vivo and in vitro. However identifying the mechanism and location of cysteine disulfide pairs remains difficult, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines during P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely sites for sulfhydryl reactivity. Point mutations at these residues have been cloned and purified. These mutants have native-like circular dichroism spectra, and similar stability to wild type in guanidine chloride. These mutants show dramatic differences in yield, kinetics, and folding intermediates from wild type. Double and triple mutants at the three C-terminal cysteine residues exhibit extremely reduced expression yields in comparison to the single mutants. The absence of both cysteines at 613 and 635 completely inhibit the ability to form trimer in vivo; double mutants at cysteines 496 and either 613 or 635 lead to barely detectable trimer levels, suggesting that there are critical interactions between these residues necessary for efficient trimer formation. The presence of external redox agents in the refolding buffer interferes with the ability of wild type tailspike to properly assemble, affecting both the overall yield of the refolding reactions and the rate at which the reaction proceeds. Interactions with monothiol redox buffer components trap a folding intermediate as a monomer, which is able to form native trimer upon reduction of the heterogeneous disulfide bond. Investigations into the folding properties of the intermediates reveal that the formation of a dimer intermediate appears to be the rate-limiting step in the C-terminal cysteine mutants, while a post-dimer reaction likely governs the wild-type trimer formation rates. Taken together, these data suggest that the non-native disulfide bond is essential for efficient assembly of the tailspike trimer.