Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible.
The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.
Gerken
Uwe
U
Institute of Microbiology, University of Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany. gerken@uni-hohenheim.de
Erhardt
Dagmar
D
Bär
Gerda
G
Ghosh
Robin
R
Kuhn
Andreas
A
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C412443:YIDC protein, E coli; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan