Engineering a Fluorescent Calmodulin for Use in Complex Models of Cardiac Physiology
Creators:Jones, Benjamin R.
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. School of Biomedical Science Honors Theses; 2009
Calmodulin (CaM) is a ubiquitously expressed calcium-binding protein that plays a critical role in cardiac physiology. CaM senses intracellular Ca2+ changes by binding the Ca2+ ion with one of its four calcium-binding loops, causing a conformational change in the CaM protein structure, and thereby affecting CaM’s affinity for its multiple intracellular protein targets. Since Ca2+ transients rise and fall in the heart on a beat-to-beat basis, an understanding of the kinetics of CaM-Ca2+ binding and dissociation events are essential for studying how cardiac systems are regulated. Isolated CaM kinetic experiments have described CaM behavior in controlled solutions, but aren’t directly applicable to the protein’s complex physiological environment. We employed site-directed mutagenesis to generate three CaM mutants with Threonine to Cysteine substitutions at residues 5, 34, and 44 (T5C, T34C, T44C). A fluorescent probe (IAANS) was covalently linked to the introduced Cysteine residue. These fluorescent Calmodulin proteins have a unique fluorescent behavior that differentiates them from any intrinsic (Tryptophan, Phenylalanine, Tyrosine) fluorescent signals. We demonstrate that two of these proteins (T5C, T34C) have fluorescent behavior that closely mimics that of WT CaM in the presence of representative peptide fragments of Myosin Light Chain Kinase (RS-20), Neural Nitric Oxide Synthase (nNOS), Calcineurin (CaN), and CaM-dependent Kinase II (CKII). Thus, T5C or T34C CaM labeled with IAANS has unique fluorescent properties without drastically affecting the behavior of the CaM protein. These fluorescent proteins give researchers the freedom to study CaM kinetic characteristics in the presence of enzymes regulated by CaM, allowing for more research with direct relevance to physiological pathways.