Determination of dimer dissociation constant of neural cadherin by fluorescence resonance energy resonance

Cadherins are calcium-dependent adhesion molecules that play a prominent role in the formation of solid tissues. Cell-cell adhesion occurs through the formation of strand-crossover dimers between identical cadherin protomers. Recent studies on neural and epithelial cadherins from our lab showed a dramatic calcium-dependent difference in their kinetics of dimer disassembly, which may have a physiologically relevant role. This motivated our interest to develop methods to study calcium-dependent dimerization of cadherins. The goal of this study is to establish protocols for and energy transfer-based technique for studying dimerization by cadherins. We report Fluorescence (Forster) Resonance Energy Transfer (FRET) experiments between donor and acceptor fluorophore-labeled proteins and the determination of Kd using the change in FRET signal as a function of protein concentration. These studies will provide the basis for a long-term interest of our lab in the geometry of the protomers at the adhesive dimerization interface.;To measure the calcium-dependent dimerization kinetics and equilibrium properties that govern the dynamics of adherent junction, we employed FRET to study adhesive dimer formation by Neural- cadherin (NCAD). We engineered Cysteine mutations that were subsequently covalently labeled with IAEDANS as donor and Alexa Fluoro 488 as acceptor fluorophores at position 87 for solution phase work.;We used two titration methods to determinate Kd for the dimerization process. First, solutions with low concentration of acceptor were titrated with solutions with high concentration of donor. Second, solutions with high concentration of donor were titrated with solutions with low concentration of acceptor. These two experiments allowed us to span a broad range of protein concentration. The midpoints of transitions indicate NCAD had an association constant (Kd = 25.0 +/- 3.0 microM) that was similar to values from the literature (Kd = 25. 8 +/- 1.5 microM; K d = 28. 1 +/- 4.0 microM).