INDEPENDENT FOLDING OF THE DOMAINS IN THE HYDROPHILIC SUBUNIT IIAB(MAN) OF THE MANNOSE TRANSPORTER OF ESCHERICHIA-COLI

The active form of the hydrophilic subunit (IIAB(man)) of the mannose transporter of Escherichia coil is a homodimer of two 35-kDa subunits. Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion. Separation of tryptic frag ments yields monomers of IIB and dimers of IIA, which are active and s table. To test whether the domains fold as independent units, the effe cts of guanidine hydrochloride (GuHCl) and temperature on the structur al stability of the intact IIAB(man) were compared with those of the i solated fragments. Equilibrium GuHCl-induced reversible unfolding, mea sured by circular dichroism and tryptophan fluorescence, showed a biph asic transition for intact IIAB(man) and monophasic transitions for ea ch isolated fragment. The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and se cond transitions of intact IIAB(man), Analytical ultracentrifugation s tudies suggested that dissociation precedes the unfolding of IIA. Ther mal unfolding of IIAB(man), monitored by differential scanning calorim etry, showed two well-separated transitions near 52 and 95 degrees C w hich corresponded to the midpoint transitions of the isolated IIB and IIA fragments. The combined results demonstrate an independent stepwis e unfolding of the domains in IIAB(man) as well as the absence of stab ilizing interdomain interactions. The lack of interdomain interactions suggests an unrestricted domain motion. This may play an important ro le in the phosphoryl transfer reaction, which is catalyzed by the bind ing of IIAB(man) to a phosphoryl carrier protein HPr (via the IIA doma in) and to the transmembrane subunits of the mannose transporter (via the IIB domain).