Control of the unidirectional topological orientation of a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c reconstituted into proteoliposomes.
Control of the unidirectional topological orientation was achieved for a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c (RC/cyt c) reconstituted into proteoliposomes. Using the method of Ueno et al. [Ueno et al. (1995) Mater. Sci. Eng. C3, 1-6], we prepared RC/cyt c by conjugating cyt c to the H-subunit of RC of Rhodobacter sphaeroides R-26 using a bifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), as previously reported. The freeze-thaw method was used to incorporate RC/cyt c into liposomes that contained dipalmitoyl-L-alpha-phosphatidylcholine and dipalmitoyl-L-alpha-phosphatidylglycerol (1:9). The topological orientation of RC/cyt c in the proteoliposomes was determined using three methods: (i) release of the cyt c moiety from the proteoliposomes by cleaving the disulfide bond in the linker residue, (ii) electron transfer from free cyt c outside the proteoliposomes to the RC moiety, and (iii) photo-induced membrane potential of RC- and RC/cyt c-reconstituted proteoliposomes. The results indicated that about 90% of the RC/cyt c in proteoliposomes was oriented with the H-subunit exposed on the outside of the liposomes, whereas only about 60% of the RC in proteoliposomes had this orientation. Thus, we successfully controlled the unidirectional topological orientation of the RC moiety in liposomes using the RC/cyt c complex.
Ueno
T
T
Department of Bioresources Chemistry, Chiba University, Matsudo, 271-0092, Japan.
Hara
M
M
Kamo
N
N
Fujii
T
T
Miyake
J
J
D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry*; D012242:Rhodobacter sphaeroides / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D045322:Photosynthetic Reaction Center Complex Proteins; D011510:Proteolipids; D013388:Succinimides; C020485:proteoliposomes; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate