Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase.

Temperature sensitive mutations fall into two general classes: tl mutations, which render the mature protein thermolabile, and tsf (temperature sensitive folding) mutations, which destabilize an intermediate in the folding pathway without altering the functions of the folded state. The molecular defects caused by tsf mutations have been intensively studied for the elongated tailspike endorhamnosidase of Salmonella phage P22. The tailspike, responsible for host cell recognition and attachment, contains a 13 strand parallel beta coil domain. A set of tsf mutants located in the beta coil domain have been shown to cause folding defects in the in vivo folding pathway for the tailspike. We report here additional data on 17 other temperature sensitive mutants which are in the beta coil domain. Using mutant proteins formed at low temperature, the essential functions of assembling on the phage head, and binding to the O-antigen lipopolysaccharide (LPS) receptor of Salmonella were examined at high temperatures. All of the mutant proteins once folded at permissive temperature, were functional at restrictive temperatures. When synthesized at restrictive temperature the mutant chains formed an early folding intermediate, but failed to reach the mature conformation, accumulating instead in the aggregated inclusion body state. Thus this set of mutants all have the temperature sensitive folding phenotype. The prevalence of tsf mutants in the parallel beta coil domain presumably reflects properties of its folding intermediates. The key property may be the tendency of the intermediate to associate off pathway to the kinetically trapped inclusion body state.