KSEF RDE: Using Calmodulin to Probe the Role of Intrinsic Disorder in Biomolecular Recognition

In recent years it has become apparent that intrinsically disordered regions (IDR5) within proteins are extremely common and often involved in important biological functions. IDRs have been found to play crucial roles in transcription, signaling pathways and immune systems in higher organisms. IDR5 are involved in numerous diseases states including cardiovascular and protein aggregation diseases. IDRs often function by undergoing a disorder to order transition when bound by another protein (i.e. they fold upon binding). It has been hypothesized that Nature has evolved disordered binding regions in order to partially decouple binding specificity from affinity. In other words, what is bound is somewhat decoupled from how strongly it is bound. An extremely important system that appears to take advantage of this disorder-mediated decoupling is calmodulin (CaM) and its binding targets (CaMBT5). Unbound CaMBT sequences are often disordered. When CaM binds, it induces, in most cases, a-helical structure in the CaMBT. We propose using this system to test the hypothesis that disorder partially decouples specificity from affinity. Under this hypothesis specificity is determined by the residues that directly contact CaM. Affinity is determined in part by the propensity for the unbound CaMBT to adopt a-helical structure. We can modulate the a-helicity of CaMBT peptides by altering residues on either side of the CaMBT, thereby altering the affinity without changing specificity. The extent to which we have altered the a-helicity of the CaMBTs is readily determined using circular dichroism spectroscopy, while binding affinity can be determined using fluorescence and/or isothermal titration calorimetry. Should the hypothesis hold, we would expect a positive correlation between the measured a-helix contents and the binding affinities. This system will serve to generate the preliminary data necessary for future studies of the reasons behind Nature's widespread use of lDRs in critical molecular recognition processes.