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- light scattering (3)
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Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein
(2001)
Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike ofSalmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel ;-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel ;-helix protein with high structural similarity to its functional homolog from phage P22.
Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro
(2010)
Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tail-spikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.
TSPs (tailspike proteins) are essential infection organelles of bacteriophage P22. Upon infection, P22TSP binds to and cleaves the O-antigen moiety of the LPS (lipopolysaccharide) of its Salmonella host To elucidate the role of TSP during infection, we have studied binding to oligosaccharides and polysaccharides of Salmonella enteric Typhimurium and Enteritidis in vitro. P22TSP is a trimeric beta-helical protein with a carbohydrate-binding site on each subunit. Octasaccharide O-antigen fragments bind to P22TSP with micromolar dissociation constants. Moreover, P22TSP is an endorhamnosidase and cleaves the host O-antigen. Catalytic residues lie at the periphery of the high-affinity binding site, which enables unproductive binding modes, resulting in slow hydrolysis. However, the role of this hydrolysis function during infection remains unclear. Binding of polysaccharide to P22TSP is of high avidity with slow dissociation rates when compared with oligosaccharides. In vivo, the infection of Salmonella with phage P22 can be completely inhibited by the addition of LPS, indicating that binding of phage to its host via TSP is an essential step for infection.
Mutations improving the folding of phage P22 tailspike protein affect its receptor binfing activity
(1999)
Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (R-S). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions.