The binding of C2 domains to phopholipid bilayers

C2 domains are responsible for the Ca²⁺-dependent binding to membranes of proteins containing the domain. The objectives of this research were:

(1) To study the Ca²⁺ dependence of the binding of C2 domains to lipid bilayers, and to determine how the Ca²⁺ affinity of the C2 domain depends on lipid structure.

(2) To study the dependence of C2 binding to lipid bilayers on lipid structure and determine to what extent properties of the lipid bilayer such as charge affects binding.

The C2 domain from PKCα was cloned, expressed and purified and a convenient procedure for refolding the C2 domain of cPLA₂ from inclusion bodies was established. A variety of fluorescence spectroscopy approached were used in this study; including changes in tryptophan fluorescence intensity, quenching of tryptophan fluorescence on binding to brominated lipids, and fluorescence energy transfer from tryptophan to lipid bilayers containing dansyl-labelled phosphatidylethanolamine. The measured affinities of the C2 domains of cPLA₂ and PKCα for Ca²⁺ do not vary with anionic lipid content and Mg²⁺ concentration as expected from calculations based on the Gouy-Chapman theory. The reason for this is probably due to the affect of competition between binding of H⁺ and Ca²⁺ to the C2 domain. H⁺ and Ca²⁺ are in competition for binding to the C2 domain, binding decreasing in affinity with decreasing pH. Thus the effect of membrane surface charge on local concentrations of H⁺ has to be considered as well as the effect on local concentrations of Ca²⁺. If 2 H⁺ bind to a Ca²⁺ binding site, in competition with binding of one Ca²⁺ ion, the effects of membrane potential cancel out so that the measured affinity for Ca²⁺ will be independent of surface charge effects. Since each Ca²⁺ binding site on a C2 domain contains ca 2 carboxyl groups it is likely that binding of Ca²⁺ to each site will indeed be in competition with binding of ca 2 H⁺ ions, this then leading to binding of Ca²⁺ independent of surface charge effects, as observed.

University of Southampton

Roscoe, Patricia Anne

2005

Roscoe, Patricia Anne

Roscoe, Patricia Anne (2005) The binding of C2 domains to phopholipid bilayers. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

C2 domains are responsible for the Ca²⁺-dependent binding to membranes of proteins containing the domain. The objectives of this research were:

(1) To study the Ca²⁺ dependence of the binding of C2 domains to lipid bilayers, and to determine how the Ca²⁺ affinity of the C2 domain depends on lipid structure.

(2) To study the dependence of C2 binding to lipid bilayers on lipid structure and determine to what extent properties of the lipid bilayer such as charge affects binding.