A REVERSIBLY UNFOLDING FRAGMENT OF P22 TAILSPIKE PROTEIN WITH NATIVE STRUCTURE - THE ISOLATED BETA-HELIX DOMAIN

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, w hich contains the known folding mutation sites, consists of three larg e right-handed parallel beta-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfoldin g at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the beta-helix. We t herefore expressed and isolated a tailspike fragment comprising only i ts central beta-helix domain (residues 109-544). As shown by equilibri um ultracentrifugation, the isolated beta-helix is a monomer at concen trations below 1 mu M and trimerizes reversibly at higher protein conc entrations. Both the similarity of fluorescence and CD spectra, compar ed 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 fo lding mutations on the thermodynamic stability of the domain and of st ructural features responsible for folding and stability of the paralle l beta-helix motif in general.