ALG-2 dimerization was studied using F?rster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined， the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25?°C， in the absence of divalent ions， the wild-type homodimer， ΔGF122 homodimer， and heterodimer display dissociation constants of 7.1， 26， and 4.5?nM， respectively. At 35?°C， subunit interaction is weaker， indicating that dimer formation is exothermic. Binding of Mg in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by... More
ALG-2 dimerization was studied using F?rster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined， the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25?°C， in the absence of divalent ions， the wild-type homodimer， ΔGF122 homodimer， and heterodimer display dissociation constants of 7.1， 26， and 4.5?nM， respectively. At 35?°C， subunit interaction is weaker， indicating that dimer formation is exothermic. Binding of Mg in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca likewise has a stabilizing effect， its direct influence on dimer stability would be negligible at cytosolic Ca levels. However， dimer stability is substantially increased by the Ca-dependent binding of ALG-2 target-peptides， suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility， the micelles ostensibly acting as target-protein surrogates. Paradoxically， however， the detergent markedly destabilizes ALG-2 dimers， particularly in the presence of Ca.
