A reversibly unfolding fragment of P22 tailspike protein with native structure: the isolated beta-helix domain

Abstract

The homotrimeric tailspike endorhamnosidase of phage P22 has been used to compare in vivo and in vitro folding pathways and the influence of single amino acid substitutions thereon. Its main structural motif, which contains the known folding mutation sites, consists of three large right-handed parallel beta-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfolding at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the beta-helix. We therefore expressed and isolated a tailspike fragment comprising only its central beta-helix domain (residues 109-544). As shown by equilibrium ultracentrifugation, the isolated beta-helix is a monomer at concentrations below 1 microM and trimerizes reversibly at higher protein concentrations. Both the similarity of fluorescence and CD spectra, compared to the complete protein, and the specific binding and hydrolysis of substrate suggest a nativelike structure. Moreover, urea denaturation transitions of the beta-helix domain are freely reversible, providing the basis for a future quantitative analysis of the effects of the folding mutations on the thermodynamic stability of the domain and of structural features responsible for folding and stability of the parallel beta-helix motif in general.