Unfolding mechanism of soluble CLIC1

Abstract

CLIC1 is an intracellular membrane protein that has an unusual property distinct from typical membrane proteins. It is able to exist in both a soluble and membrane-bound form in cells. The membrane-insertion mechanism of soluble CLIC1 is still unknown. However, it has been proposed that soluble CLIC1 has to undergo unfolding and refolding processes to form its membrane-bound conformation. In this study, the focus is on the understanding of the unfolding mechanism of soluble CLIC1 under the pH values typical of the cytosol and the membrane surface. Equilibrium unfolding studies show that, at pH 7.0, CLIC1 unfolds via a two-state transition and the unfolding kinetics studies further confirms that there are no detectable transient intermediates. At pH 5.5, CLIC1 shows a three-state unfolding transition with a formation of an intermediate species at equilibrium in the presence of 3 ~ 4 M urea. The characterisation of this intermediate indicates that it has reduced secondary structural content and native-like tertiary structure with the exception of a more buried Trp35 when compared to the native state. Moreover, this intermediate has more exposed hydrophobic surface than the native state. The unfolding kinetics studies demonstrate that there are hidden intermediates in the native-to-unfolded transition at pH 5.5. The formation of the intermediate state involves, initially, a rapid partial unfolding followed by a slow repacking of the structure. The results from equilibrium unfolding and unfolding kinetics studies both suggest that the intermediate state is more stable than the native state at pH 5.5, indicating that under this condition, the stability of native CLIC1 is reduced and the intermediate is energetically favoured. The results from this study give information about the conformational stability and the different unfolding behaviours of soluble CLIC1 under the conditions that simulate the environments of the cytosol and the membrane surface, and further provide a possible interpretation for the in vivo membrane-insertion mechanism of the protein.