A giant liposome for single-molecule observation of conformational changes in membrane proteins.
We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F(O)F(1)) in liposomes tens of mum in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.
Department of Functional Molecular Science, The Graduate University for Advanced Studies (Sokendai), Okazaki, Aichi 444-8585, Japan.
Yasuhiro
Y
Onoue
Toshiharu
T
Suzuki
Max
M
Davidson
Mattias
M
Karlsson
Owe
O
Orwar
Masasuke
M
Yoshida
Kazuhiko
K
Kinosita
D000255:Adenosine Triphosphate; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008857:Microscopy, Interference; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry; D012399:Rotation
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D010455:Peptides; D011510:Proteolipids; D000255:Adenosine Triphosphate