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 beta-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 (Nagar et al., Nature 1995;380:360-364). Recently, stability studies of ECAD2 (Prasad et al., Biochemistry 2004;43:8055-8066) were undertaken with the motivation that Domain 2 is a representative domain for this family of proteins. The definition of a domain boundary is somewhat arbitrary; hence, it was important to examine the effect of the adjoining linker regions that connect Domain 2 to the adjacent domains. Present studies employ temperature-denaturation and proteolytic susceptibility to provide insight into the impact of these linkers on Domain 2. The significant findings of our present study are threefold. 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, 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. Thus, the context of Domain 2 within the whole molecule affects its thermodynamic characteristics. Study holds ProTherm entries: 22049, 22050, 22051, 22052, 22053, 22054, 22055, 22056, 22057, 22058, 22059, 22060, 22061, 22062, 22063, 22064 Extra Details: domain 2 (residues 107-212) thermodynamics; calcium binding; modular domains; circular dichroism; thermal denaturation
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:53 p.m.
|Number of data points||32|
|Proteins||Cadherin-2 ; Cadherin-2|
|Assays/Quantities/Protocols||Experimental Assay: dCp ionic:NaCl,Ca2+: 140 mM,5 mM, temp:25.0 C ; Experimental Assay: dG ionic:NaCl,Ca2+: 140 mM,5 mM ; Experimental Assay: dCp ionic:NaCl,Ca2+: 140 mM,5 mM ; Experimental Assay: Tm ionic:NaCl,Ca2+: 140 mM,5 mM ; Experimental Assay: dCp temp:25.0 C, ionic:NaCl: 140 mM ; Experimental Assay: dG ionic:NaCl: 140 mM ; Experimental Assay: dCp ionic:NaCl: 140 mM ; Experimental Assay: Tm ionic:NaCl: 140 mM|
|Libraries||Mutations for sequence GSDWVIPPINLPENSRGPFPQELVRIRSGRDKNLSLRYSVTGPGADQPPTGIFIINPISGQLSVTKPLDRELIARFHLRAHAVDINGNQVENPIDIVINVIDMNDNRPEF|