Preparation and characterisation of soluble CLIC1 and liposomes
Date
2009-07-07T11:42:41Z
Authors
Adamson, Roslin Jane
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
CLIC1 is an intracellular membrane protein that has the intriguing property of
being able to exist in both soluble and integral membrane forms. It is unknown
how CLIC1 converts from a soluble to a membrane-inserted conformation, but it
has been proposed that the transition involves the unfolding of certain regions of
the protein followed by refolding into a membrane-competent form. This study
characterised the structure and stability of reduced, soluble CLIC1 at the pH
values it would encounter in both bulk cytosol and at the membrane surface.
Additionally, the preparation and properties of a model membrane system were
characterised. At pH 7.0 CLIC1 is more stable and follows a cooperative twostate
unfolding transition with a G(H2O) of 10.3 kcal/mol and m-value of 2.3
kcal/mol M-1 urea. At pH 5.5 the CLIC1 native structure is looser and more
flexible with lower secondary structural content, is less stable, and unfolds via a
stable intermediate with exposed hydrophobic surfaces. The G(H2O) and mvalues
for formation and unfolding of this species are well into the dimeric range,
and data from the local probe, Trp35, indicate that the intermediate may be
oligomeric. The existence of the intermediate species at low pH and under mild
denaturing conditions suggests a mechanism whereby CLIC1 may form channels
in vivo. Anionic large unilamellar vesicles prepared with a 4:1:1 molar ratio of
phosphatidylethanolamine, phosphatidylserine and cholesterol, respectively, were
stable up to approximately 50 °C and were highly reproducibly and
homogenously sized at ~200 nm diameter. Basal leakage of encapsulated
chloride-sensitive fluorescent dye at room temperature was modest for two to
three days, and was minimal for up to seven days at 4 °C. These vesicles should
prove to be an ideal membrane system for the study of membrane-inserted CLIC1
and with encapsulation of a chloride-sensitive dye may provide the means for a
viable functional assay for CLIC1 channel activity.