Single Molecule Take-and-Place Technique for Positioning a Membrane Protein on a Lipid Bilayer

Abstract

Manipulating a single protein molecule on liposomes or planar lipid bilayers is a useful technique for studying membrane-bound proteins, receptors, or ion transporters and for delicate applications including biosensor chips, drug screening, and clinical diagnoses. However, several key breakthroughs are required for success through difficult techniques such as single protein take-and-place with reasonable spatiotemporal control. In this report, a novel method was established for placing a single transmembrane protein onto a patterned lipid bilayer. A surface-functionalized 1.8 nm gold nanoparticle was first engineered at the tip of an atomic force microscope. A single transmembrane protein, H+-translocating pyrophosphatase with six-histidine residues tag, was then picked up using such a microscopy tip where its nanoparticle was modified by nickel-nitrilotriacetic acid. Two-step fluorescence bleaching observation and quantum dot blinking analysis subsequently verified successful manipulation of a single functional protein on a lipid membrane in a predetermined manner. Furthermore, the enzymatic activity from the single H+-translocating pyrophosphatase was determined, demonstrating that membrane proteins retain their functions on the lipid bilayer through a single-molecule take-and-place technique. This innovative technique overcomes current limitations and provides a single biomolecule nanomanipulation system for versatile studies of membrane-bound proteins.