Unfolding of bacteriophage P22 tailspike protein: enhanced thermal stability of an N‐terminal fusion mutant

Abstract

The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor. Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation. We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end-terminal peptide fusions. Whereas the C-terminal fusion has minor effects on the TSP stability, the presence of a 23-mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process. At 65³C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3U10 3R s 3I for ATSP versus 1.7U10 3Q s 3I for wild-type TSP. On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide. These results suggest a critical participation of the N-terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.z 1998 Federation of European Biochemical Societies.

Ɛ34 Phage Tailspike Protein is Resistant to Trypsin and Inhibits and Salmonella Biofilm Formation

“…P22 TSP was also treated with trypsin in the presence of 2% SDS, migrated at 126 KDa size, which corresponds to 215 KDa size of the P22 TSP trimer in actual calculated molecular weight. Similar reports of migration characteristics of the trimeric protein of P22 in a 7.5% polyacrylamide gel has been published by X. Carbonell and A. Villaverde, indicating a 126±3 KDa migration in gel but an actual size of 215 KDa [57]. As depicted in Figure 3B, EEUTUH (the extended Ɛ34 TSP untreated and unheated) recorded the trimeric species migrating at blurry consistency at averagely 161 KDa.…”

“…P22 TSP was also treated with trypsin as shown in eighth lane of same Figure 3B. A single band was observed at 126 KDa, which corresponds to 215 KDa size of the P22 TSP trimer in actual calculated molecular weight similar to reports by Carbonell and Villaverde [58].…”

Protein aggregation as bacterial inclusion bodies is reversible

“…TSP is a homotrimeric protein that undergoes a complex folding pathway from which unfolded intermediates collapse as IBs under overproduction conditions [17,18]. In addition, TSP has been extensively used as a model for the analysis of molecular interactions between aggregated polypeptides [19] and of protein folding and unfolding pathways [20–24]. The arrest of recombinant protein synthesis in IB‐carrying cells results, as in the case of β‐galactosidase, in IB disintegration (Fig.…”