6533b7d0fe1ef96bd125a1d3

RESEARCH PRODUCT

Incorporation of membrane proteins into lipid surface monolayers: Characterization by fluorescence and electron microscopies

subject

description

The preparation of oriented protein samples is an attractive goal, e.g., to gain more detailed information from spectroscopic experiments. Our approach towards this aim was to prepare monolayers of phospholipids at the air-water interface and to incorporate the proteins into these ordered structures. Subsequently, we used the Langmuir-Boldgett (LB) transfer technique to obtain samples of oriented proteins on solid supports. — Incorporation was achieved by spreading the proteins from a detergent solution onto a prespread lipid monolayer on the water surface. We characterized successful incorporation by in situ fluoresence microscopy and by electron microscopy, and investigated the topology of transferred monolayers. — Our results show that the choice of the lipid matrix is pivotal. We present an optimized lipid matrix system that facilitates protein incorporation, while enabling LB transfer at the same time. — Fluorescence microscopic investigations revealed that the lateral phase structure of the lipid films remains qualitatively unaffected by the reconstitution, with the proteins incorporated into the fluid phases. Quantitative evaluations of the protein content of the thin film samples indicated that ∼15% of the proteins spread were incorporated, whereas evaluation of the additional area of incorporation (of protein and detergent) showed that a substantial amount of detergent was incorporated as well.