The Metal-Ion Binding Properties and Target-Binding Abilities of Helix-Loop-Helix EF-hand Proteins
Date
2013-01-08
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Abstract
The EF-hand calcium-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by the helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the calcium ion, and the conformational change that accompanies calcium-binding to these sites facilitates calcium-dependent signalling. In this thesis, two critical characteristics pertaining to this class of proteins are examined. First, their ability to bind the activating calcium ion is studied using various biophysical techniques. The calcium-binding properties of human calcium- and integrin-binding protein 1 and two soybean calmodulin isoforms (one conserved and one divergent) were examined as well as the ability of these proteins to select against the chemically similar magnesium cation. Not unexpectedly, our results point to an intimate link between the calcium affinity of a given EF-hand and the physiological role of the protein. Second, the structural details of calcium-dependent intermolecular interactions between the cytosolic human EF-hand protein calmodulin and three known targets were determined in solution using nuclear magnetic resonance spectroscopy. Central to this research was the exploitation of calmodulin’s critical methionine residues to serve as probes for these interactions. The development of a novel methodology is described through which the solution structures of calcium-calmodulin:peptide complexes can be derived from a limited set of experimental restraints. This approach is then applied to study at a structural level the regulatory relationship between calmodulin and the leukocyte homing receptor L-selectin. Calmodulin inhibits the proteolysis of L-selectin’s extracellular domains through an unknown mechanism critical to understanding leukocyte trafficking. Our solution structure and accompanying biophysical study provide the first molecular insight into the emerging new role for calmodulin as a transmembrane signalling protein. Finally, research on one of the largest calcium-calmodulin:target complexes studied to date is presented, that between calmodulin and the intact immuno-protein lactoferrin. Taken together, this thesis serves to expand our knowledge in the field of eukaryotic EF-hand proteins as well as present experimental approaches for answering common questions in this area.
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Biochemistry
Citation
Gifford, J. (2013). The Metal-Ion Binding Properties and Target-Binding Abilities of Helix-Loop-Helix EF-hand Proteins (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/28248