Research

Calmodulin (CaM) is a small Ca2+-binding protein that binds to and regulates a number of different protein targets. Its ability to tightly bind to a diverse set of small peptides makes it an ideal system for searching for new binding targets. Hundreds of sequences are already known to bind CaM, leading to suggestions regarding the basic elements required for binding. Several high-resolution CaM-peptide complexes have also been solved by X-ray crystallography. Further, the ORBIT computational design software was successfully used to generate a CaM variant with increased specificity toward a particular target. Using this abundant knowledge of CaM binding affinity and specificity, we intend to design libraries of CaM variants to bind novel peptides with high affinity. To be able to experimentally validate the libraries, we first need to develop a high-throughput assay to determine CaM-peptide binding. Förster resonance energy transfer (FRET) is a state-of-the-art method to characterize interactions between molecules. Color variants of green fluorescent protein (GFP) can be attached to a host protein (CaM), and FRET can be used to determine protein-protein interactions in vivo, thus allowing identification of CaM-binding peptides. Using a 96-well plate-based assay, we expect high-number libraries can be tested experimentally and novel targets can be found efficiently.


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