Bilayer mediated nonspecific modification of MscL function by
phytochemicals
BSc. research project proposal
Project supervisors:
Keywords:
H.I. Ingólfsson & A. Koçer
[email protected], phone: 2797, room 5115.0005
plant phenols, modulation of bilayer properties, mechanosensitive channel of
large conductance
Introduction:
Biologically active plant phenols (phytochemicals) have been a cornerstone of traditional medicine
around the world. Recently these agents have gained widespread popularity in Western medicine, to
the extent that thousands of scientific papers are published on their activity each year. These
compounds exert a broad range of pharmacological effects including antioxidant, anti-inflammatory,
anticarcinogenic and antimicrobial effects, but the mechanism(s) of action is usually ill-defined.
The better studied compounds modulate the function of a multitude of unrelated proteins, with very
few having identified binding sites. Yet, different phenolic compounds often affect the same proteins,
many of which are membrane-associated (Fig. 2). Moreover a given compound usually alters the
different proteins at similar concentrations. Additionally, many bioactive phytochemicals are
hydrophobic/amphipathic and tend to adsorb to lipid bilayers. Also, in spite of large variations in
chemical structure among these phenolic compounds (Fig. 1), plant phenols not infrequently have
synergistic effects.
The lipid bilayer serves as a gate-keeper/regulator for many cell functions, and a phytochemical
induced modulation of bilayer properties can explain, at least partially, their unspecific mode of
action(s). We therefore measured their changes to bilayer properties using gramicidin A channels as
probe and found all the tested phytochemicals to affect bilayer material properties and at
concentrations consistent with their reported biological activity.
Aim:
The aim of this project is to verify the phytochemicals effect on membrane properties by testing if they
can alter the function of another membrane protein that also is sensitive to changes in membrane
properties. We proposed to test the effect of five heavily studied phytochemicals (see Fig. 1) on the
function of the mechanosensitive channel of large conductance (MscL). The MscL serves as a last
resort emergency release valve to protect bacteria from lysis upon acute osmotic shock. When the
bacterial membrane swells the induced bilayer tension activates the MscL. This tight bilayer coupling
makes this MscL an ideal system to test for changes in bilayer properties.
In this project the student will gain experience in the following techniques:
1. Isolation of a membrane protein by using affinity chromatography
2. Reconstitution of an isolated membrane protein into synthetic lipid bilayers
3. Encapsulating a fluorescent dye into the proteoliposomes (product of step 2)
4. Analyse the effect of the components on the channel activity by using a fluorescent
dequenching assay
5. Learn to analyse the results
Figure 1: Structure
of the
phytochemicals:
capsaicin, curcumin,
EGCG, genistein
and resveratrol.
Capsaicin
EGCG
Curcumin
Genistein
Resveratrol
Figure 2: Membrane
protein reported to be
affected by curcumin,
EGCG, resveratrol,
genistein or capsaicin.
(+) indicates
activation or upregulation. (-)
indicates inhibition or
down-regulation. (*)
indicates
“interaction”. (±)
indicates biphasic
dose response curve.
References are not
listed due to space
limitations.
Literature:
• Ingolfsson HI, Koeppe RE 2nd, Andersen OS. Curcumin is a modulator of bilayer material
properties. Biochemistry. 2007;46:10384-91.
• Koçer A, Walko M, Feringa BL. Synthesis and utilization of reversible and irreversible lightactivated nanovalves derived from the channel protein MscL. Nat Protoc. 2007;2:1426-37.