Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that trans ports iron coupled to ferrichrome and is the receptor for a number of bacte riophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta -barrel lodged in the membrane, extracellular hydrop hilic loops, and a globular domain (the "cork") located within the beta -ba rrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the di fferent functions of the protein. To address these questions, we have compa red the properties of the wild-type receptor to those of a mutated FhuA (Fh uA Delta) missing a large part of the cork. Differential scanning calorimet ry experiments on wild-type FhuA indicated that the cork and the beta -barr el behave as autonomous domains that unfold at 65 and 75 degreesC, respecti vely. Ferrichrome had a strong stabilizing effect on the loops and cork sin ce it shifted the first transition to 71.4 degreesC. Removal of the cork de stabilized the protein since a unique transition at 61.6 degreesC was obser ved even in the presence of ferrichrome. FhuA Delta showed an increased sen sitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.