Fourier transform infrared spectroscopy study of the secondary and tertiary structure of the reconstituted Na+/Ca2+ exchanger 70-kDa polypeptide.
The secondary structure of the purified 70-kDa protein Na+/Ca2+ exchanger, functionally reconstituted into asolectin lipid vesicles, was examined by Fourier transform infrared attenuated total reflection spectroscopy. Fourier transform infrared attenuated total reflection spectroscopy provided evidence that the protein is composed of 44% alpha-helices, 25% beta-sheets, 16% beta-turns, and 15% random structures, notably the proportion of alpha-helices is greater than that corresponding to the transmembrane domains predicted by exchanger hydropathy profile. Polarized infrared spectroscopy showed that the orientation of helices is almost perpendicular to the membrane. Tertiary structure modifications, induced by addition of Ca2+, were evaluated by deuterium/hydrogen exchange kinetic measurements for the reconstituted exchanger. This approach was previously proven as a useful tool for detection of tertiary structure modifications induced by an interaction between a protein and its specific ligand. Deuterium/hydrogen exchange kinetic measurements indicated that, in the absence of Ca2+, a large fraction of the protein (40%) is inaccessible to solvent. Addition of Ca2+ increased to 55% the inaccessibility to solvent, representing a major conformational change characterized by the shielding of at least 93 amino acids.
Saba
R I
RI
Laboratoire de Pharmacodynamie et de Thérapeutique, Faculté de Médecine, Bât. GE, 808 route de Lennik, B-1070, Brussels, Belgium.
Ruysschaert
J M
JM
Herchuelz
A
A
Goormaghtigh
E
E
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003903:Deuterium; D006352:Heart Ventricles / Q000378:metabolism; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared
D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger; C020485:proteoliposomes; C024990:asolectin; D003903:Deuterium; D002118:Calcium