TRYPTOPHAN-CONTAINING ALPHA-SUBUNITS OF THE ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE - ENZYMATIC AND UREA STABILITY PROPERTIES

Early studies suggested that the Escherichia coli tryptophan synthase alpha-subunit unfolded in a two-step process in which there was a stab le intermediate composed of a native alpha-1 folding unit (residues 1- 188) and a completely unfolded alpha-2 folding unit (residues 189-268) . More recent evidence has indicated that such a structure for the int ermediate seems unlikely. In this report, single Trp residues (absent in the wild-type alpha-subunit) are substituted separately for Phe res idues at positions 139 (in alpha-1) and 258 (in alpha-2) to produce th e F139W, F258W, and F139W/F258W mutant alpha-subunits. The UV absorban ce and fluorescence properties of the F139W/F258W double mutant are id entical with those of equimolar mixtures of the single mutants, sugges ting that the Trp residue at each position can independently report th e behavior of its respective folding unit. Each mutant alpha-subunit i s wild-type enzymatically, and when UV absorbance is monitored, the ur ea induced unfolding of the three tryptophan-containing alpha-subunits is virtually identical to the wild-type protein. These wild-type prop erties make these proteins attractive candidates for a fluorescence ex amination of the behavior of the individual folding units and the stru cture of potential intermediate(s) and as host proteins for the insert ion of our existing destabilizing and/or stabilizing mutational altera tions.