Secondary and tertiary structure changes of reconstituted P-glycoprotein. A Fourier transform attenuated total reflection infrared spectroscopy analysis.
The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.
Sonveaux
N
N
Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Brussels, Belgium.
Shapiro
A B
AB
Goormaghtigh
E
E
Ling
V
V
Ruysschaert
J M
JM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D003630:Daunorubicin / Q000378:metabolism; D003903:Deuterium; D007700:Kinetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011510:Proteolipids; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D003903:Deuterium; D003630:Daunorubicin