| Epithelial cadherin is a transmembrane protein that is essential in calcium-dependent cell-cell recognition and adhesion. It contains five independently folded globular domains in its extracellular region. Each domain has a seven strand β-sheet immunoglobulin fold. Short, seven residue peptide segments connect the globular domains and provide oxygens to chelate calcium ions at the interface between the domains. It undergoes calcium-dependent self association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions). Different high resolution structural studies give conflicting views regarding the nature of cis and trans-interactions. From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory. Hence, we applied a thermodynamic approach to study the linkage between the sequential events of domain folding, Ca2+ binding and cis -interactions. The objective of this thesis is to study the energetics of domain folding of E-cadherin, a primary step in the successive events leading to higher order homophilic associations of the molecule. We have simplified the approach by first examining participating extracellular Domain2 individually and then in combination with the linkers and Domain1. Stability studies of Domain2 of E-cadherin (ECAD2) give a unified picture of the energetics of ECAD2-folding and stability, for which ΔG° is 6.6 kcal/mol, T m is 54°C, ΔHm is 90 kcal/mol, and ΔCp is 1300 cal/Kmol. The stability studies on Domain2 with linkers show the following points. First the linker segments destabilize the core domain in the absence of calcium. Second, the destabilization due to addition of the linker segments can be partially reversed by the addition of calcium. Third, addition of sodium chloride stabilizes all constructs. This result implies that electrostatic repulsion is a contributor to destabilization of the core domain by addition of the linkers. Distinct calcium stabilization is observed as a shift in T m from 40 (apo) to 65°C (10 mM Ca2+) for the two domain construct MECAD12. Spectroscopic experiments agree well with calorimetric (DSC). In the absence of calcium, the unfolding transition is shallow ((ΔH m) 40 kcal/mol) but not obviously three state. Model-dependent analysis indicates that a second transition could be assigned to the unfolding of Domain2. A calcium-binding constant was derived from the calcium-dependent shift in temperature denaturation profiles. The Kd that is obtained (55 μM) is consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calcium bound states. Hence these studies show that in spite of its modular structure, the E-cadherin molecule function energetically as a co-operative unit. |
