Regioselective photolabeling of glycoprotein A in membranes.
We have developed a chemical method for directly identifying the amino acid residues of the transmembrane domain of a protein that are located right in the center of the membrane. Glycophorin A (GPA), the major sialoglycoprotein of human erythrocytes, was the first membrane protein whose primary sequence was elucidated, but its three-dimensional structure is still not known. GPA has been reconstituted into liposomes formed from dimyristoylphosphatidylcholine, dimyristoylphosphatidylserine, cholesterol, and a bola-amphiphilic phospholipidic photoactivatable probe (radioactive probe 1) by a detergent-mediated method. Electron microscopy confirmed the formation of spherical vesicular structures, and sucrose-density gradients revealed that the proteoliposomes comprised only one membrane fraction. Proteinase-K digestion of GPA in the proteoliposomes suggested that the orientation of GPA in reconstituted proteoliposomes was virtually identical to that observed in natural erythrocyte membranes. After photo-irradiation of the reconstituted proteoliposomes and in situ tryptic digestion, the photolabeled amino acid residues were analyzed by Edman degradation and their radioactivity was measured. Val80 and Met81, which had been assumed to be located near the center of the transmembrane domain of GPA, were indeed highly selectively photolabeled by probe 1. The new method might be applied to analyze the three-dimensional arrangement of the transmembrane domain of protein complexes that are made up from several subunits.
Ogawa
Yoshikatsu
Y
Laboratoire de Chimie Organique des Substances Naturelles, Associé au CNRS, Centre de Neurochimie, Université Louis Pasteur, Strasbourg, France.
Hahn
Wolfgang
W
Garnier
Philippe
P
Higashi
Nobuaki
N
Massotte
Dominique
D
Metz-Boutigue
Marie-Hélène
MH
Rousseau
Bernard
B
Kodaka
Masato
M
Sunamoto
Junzo
J
Ourisson
Guy
G
Nakatani
Yoichi
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D006021:Glycophorins / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000032:analysis; D019920:Photoaffinity Labels
D000596:Amino Acids; D006021:Glycophorins; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D010743:Phospholipids; D019920:Photoaffinity Labels; D002784:Cholesterol