Characterization of Fas receptor peptides with calmodulin and its derivatives (969.2)

Abstract

The Fas receptor (FasR)‐mediated apoptotic pathway has been known to be regulated by Ca2+ fluctuations in the cell, specifically through Calmodulin (CaM). CaM contains four EF‐hand motifs, and upon Ca2+ binding, a conformational change is elicited, allowing FasR binding. Here we sought out to perform a characterization using synthetic Fas peptides and recombinant CaM and Troponin C chimera proteins (1TnC, 2TnC, 3TnC, and 4TnC) in which the EF‐hands have been interchanged (i.e. 1TnC contains the first EF‐hand motif of TnC and the 2nd, 3rd and 4th EF‐hands of CaM). Using isothermal titration calorimetry, we determined that a peptide with sequence TLSQVKGFVRKNGVNE binds weakly to CaM (Kd =1.8 x 10‐5 M) whereas a longer peptide (KYITTIAGVMTLSQVKGFVRKNGV) binds moderately to CaM (Kd = 2.6 x 10‐6 M). The replacement of the 1st or 2nd EF‐hands did not significantly alter the peptide binding affinity in comparison to CaM, being driven by both positive entropy and negative enthalpy changes, whereas the replacement of the 3rd and 4th EF‐hands exhibited a weaker, highly enthalpic binding. To understand the binding mechanism related to the conformational change of CaM and chimeras upon ligand binding, we are currently determining their heat capacity changes by ITC and thermostability by differential scanning calorimetry.Grant Funding Source: Supported by National Science Foundation