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 transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.
Bonhivers
M
M
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université Paris Sud, Bât 430, 91 405 Orsay Cedex, France.
Desmadril
M
M
Moeck
G S
GS
Boulanger
P
P
Colomer-Pallas
A
A
Letellier
L
L
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000378:metabolism; D006358:Hot Temperature; D008024:Ligands; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011991:Receptors, Virus / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013604:T-Phages / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008024:Ligands; D011991:Receptors, Virus; D011994:Recombinant Proteins; D005291:Ferrichrome; D012967:Sodium Dodecyl Sulfate