Biochimica et Biophysica Acta (BBA) - Biomembranes
Volume 1788, Issue 6, June 2009, Pages 1332-1340



doi:10.1016/j.bbamem.2009.01.015    

A giant liposome for single-molecule observation of conformational changes in membrane proteins

Yasuhiro Onouea, b, Toshiharu Suzukic, d, Max Davidsone, Mattias Karlssone, Owe Orware, Masasuke Yoshidac, d and Kazuhiko Kinosita Jr.b, *

aDepartment of Functional Molecular Science, The Graduate University for Advanced Studies (Sokendai), Okazaki, Aichi 444-8585, Japan

bDepartment of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

cATP Synthesis Regulation Project, ICORP, Japan Science and Technology Corporation, Aomi 2-41, Tokyo 135-0064, Japan

dChemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda 4259, Yokohama 226-8503, Japan

eDepartment of Chemical and Biological Engineering, Microtechnology Centre, Chalmers University of Technology, SE-412 96, Göteborg, Sweden


Received 10 December 2008; 
revised 25 January 2009; 
accepted 28 January 2009. 
Available online 3 February 2009.

Abstract

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 (FOF1) in liposomes tens of μm 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.

Keywords: Single-molecule; Giant liposome; Membrane protein; Conformational change; ATP synthase

*Corresponding author. Tel.: +81 3 5952 5871; fax: +81 3 5952 5877.