Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Abstract

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.Keywords: P22 tailspike protein (TSP); transient disulfide bond; assembly; disulfide shuffling; SDS P22 tailspike protein is a member of the elongated b-helix family of viral adhesins that participates in polysaccharide binding during viral infection that includes pectate lyase C and pertactin (Mitraki et al. 2002;Simkovsky and King 2006). Tailspike is a homotrimer and each monomer has 666 amino acid residues. Tailspike has four major domains: the N-terminal head-binding domain; the b-helix domain, which is formed by alternating b strands and turns; a bsheet prism domain, where three chains are interdigitated; and the C-terminal domain (Steinbacher et al. 1994(Steinbacher et al. , 1997. Each chain in the native trimer has eight reduced cysteines: six are located in the b-helix domain and two are in the Cterminal domain. Disulfide bonds are formed during tailspike assembly, and they are believed to play critical roles in the registration of the three tailspike chains (Robinson and King 1997;Haase-Pettingell et al. 2001;Danek andRobinson 2003, 2004). Our current model is that intermolecular disulfide bonds form between the C613 on one chain and the C635 on another chain in the C-terminal domain to help align the three subunits. Site-directed mutagenesis of either C613 or C635 significantly decreased the assembly kinetics of tailspike as well as final yields both in vivo and in vitro (Haase-Pettingell et al. 2001;Danek and Robinson 2003). Since the intermolecular disulfide bonds are absent in the native trimer, reduction of those disulfide bonds is a required step in tailspike folding. How does the reduction occur? The nascent chains fold on the ribosome while translation is in progress, and one could imagine many sources of reductants (Brunschier et al. 1993;Gordon et al. 1994;Sather and King 1994;Clark and King 2001). However, in vitro, purified tailspike manages to fold into native trimer in the absence of redox buffers to yields of 80%-90% (Danek and Robinson 2004). A further troubling issue has been the presence of only two detectable bands on SDS-PAGE-the monomer and the trimer-although nondenaturing P...

S-adenosylhomocysteine hydrolase (AHCY) deficiency: Two novel mutations with lethal outcome

“…However, in the presence of reducing agent DTT (100 mM), a single band appears on the gel, migrating at an almost identical position as the wild-type tetramer. Similar results for the effect of reducing agent DTT on the dissociation of intermolecular disulfide bonds have been shown by Kim and Robinson during their studies of P22 tailspike protein [Kim and Robinson, 2006]. We suppose that during native-PAGE the shift in temperature caused by electrode heat leads to changes in the dissociation constants of the buffer with a decrease in [H+].…”

“…Additionally, purity and electrophoretic behavior of the recombinant AHCY protein was analyzed using native polyacrylamide gel electrophoresis (native PAGE) as described previously [Belužić et al, 2006] and according to Kim and Robinson [2006] with reducing agent DTT. The molecular weights of recombinant mutant and wild-type forms of AHCY were analyzed by gel filtration chromatography according to Belužić and coworkers [2006].…”

Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling

“…Such temporary disulfide bonds are not without precedence as early transient disulfide-dependent steps have been shown in influenza virus nucleocapsid (51) and Simian Virus 40 maturation (52). Additionally, transient interchain disulfide bonds in the P22 tailspike adhesion proteins have been seen (53).…”

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

“…To determine disulfide bond formation, r␤-galactosidase (135-kDa) samples in either 0.25 M Tris-HCl-87% glycerol-6 mg bromophenol blue and distilled water or a 20 mM DTT buffer (25) were incubated at 37°C for 15 min and were loaded onto a nondenaturing 6% polyacrylamide gel for Western blot determination as described above. Three replicas were loaded onto each gel (25), and samples A and B were run in the same gel under the same exact conditions. As a reference (Cϩ), we used a commercial E. coli beta-galactosidase with a molecular mass of 116 kDa.…”