The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli 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 fragments yields monomers of IIB and dimers of IIA, which are active and stable. To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments. Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each 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 second transitions of intact IIABman. Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA. Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments. The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions. The lack of interdomain interactions suggests an unrestricted domain motion. This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain). Study holds ProTherm entries: 4804, 4805, 4806 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:27 p.m.
|Number of data points||4|
|Proteins||PTS system mannose-specific EIIAB component ; PTS system mannose-specific EIIAB component|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dHvH ; Experimental Assay: dG|
|Libraries||Mutations for sequence TIAIVIGTHGWAAEQLLKTAEMLLGEQENVGWIDFVPGENAETLIEKYNAQLAKLDTTKGVLFLVDTWGGSPFNAASRIVVDKEHYEVIAGVNIPMLVETLMARDDDPSFDELVALAVETGREGVKALKAKPFAG|