Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Supported lipid membranes for reconstitution of membrane proteins
3.2. VESICLE FUSION
Langmuir-Blodgett techniques are in most cases not suitable for simultaneous
deposition of lipid monolayers and integral proteins. The method has been applied
successfully only with small ionophores and integral proteins that only span half the
cell membrane as in the articles cited in the previous section. The obvious reason is that
spreading mixtures of large integral proteins and phospholipids at the air/water
interface imposes a risk for denaturation of the protein.
An alternative and very attractive method was presented by Brian and McConnell,
comprising the incubation of small unilamellar vesicles, SUV, with alkylated or
unalkylated glass coverslips to form planar membranes [58]. Lateral diffusion
coefficients for the phospholipids were obtained from fluorescence recovery after
photobleaching and showed that the membrane was fluid. When the glass slides were
incubated with vesicles containing the transmembrane protein H-2K k a planar
membrane was formed on the surface where the reconstituted protein clearly was
recognised by precursor cytotoxic T cells. However, the integral membrane protein did
not show any measurable mobility which indicates that the protein interacts strongly
with the supporting substrate. Thus, a facile way to investigate immunological
responses utilising supported lipid bilayer membranes was demonstrated for the first
time. However, a qualitative evaluation could not be carried out since the protein was
immobilised. The paper soon acquired successors and the progress of the work is
discussed in the forthcoming sections.
3.2.1. LB/hesicle method and/or direct fusion
3.2. I. 1. Structure, fluidity and formation of s-BLMs. A monolayer of dipalmitoyl
phosphatidic acid was transferred to an ATR-plate head-down with LB-techniques and
was subsequently incubated with a SUV solution. The formation of a planar bilayer
membrane and the subsequent adsorption of a local anaesthetic, oxybutylprocaine were
monitored in situ with Attenuated Total Reflection, ATR Spectroscopy [59]. It was
concluded that the adsorption of the anaesthetic did not involve significant penetration,
because of the small lipid loss, 10%, upon desorption. In later work the same group
describes the structure and stability as determined with FTIR-ATR of the same type of
supported bilayer. The mean molecular area of the phospholipids, the hydrocarbon
chain order, and in some cases the chain tilt were reported [60].
In an original paper on a new application of neutron reflectivity, SUVs were
prepared from dimyristoylphosphatidylcohline with or without perdeuterated
hydrocarbon chains. These were fused on quartz surfaces to form stable bilayer
membranes. Specular reflection of neutrons was used for the first time to investigate the
structure of a single lipid bilayer. Selective deuteration of the lipids provided the
necessary conditions for an estimation of the thickness of the total bilayer, the head
group and the hydrocarbon chain. In addition, the chain tilt angle and the degree of
lipid hydration could be calculated and the presence of a thin water film between the
lipid membrane and the quartz surface was verified and its thickness was determined to
30 + _10Å [61].
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