Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Neuroscience; Molecular, Cellular, and Developmental Biology;
Kristen M. Johansen
This thesis describes the structural and molecular analysis of two leech neuronal proteins, Calsensin and Filamin. Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons which fasciculate into a single tract in the leech central nervous system. Calsensin is a 9 kD protein with two EF-hand calcium-binding motifs. Calsensin can multimerize via disulfide bridge formation involving the two cysteine residues in vitro. Furthermore, Calsensin shows differential gel migration depending on the presence or absence of calcium suggesting a conformational change upon calcium binding. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium-bound Calsensin. Calsensin consists of four helices forming a unicornate-type of four-helix bundle and we show by analysis of EDTA and Ca2+ titrations that Ca2+ binding leads to conformation change in the protein. The backbone dynamics of the protein was determined by measuring the 15N relaxation rates and heteronuclear NOE. The internal dynamics of the protein correlates well with the three-dimensional structure and provides insight into plausible mechanisms of calcium and target protein binding. To search for putative molecular interacting partners of Calsensin, we conducted a GST-Calsensin overlay screen of leech expression library. A probable candidate is leech homolog of Caldesmon, which interacts with Calsensin in a calcium-dependent manner. We have mapped the binding surface of this interaction using HSQC experiments.;The Laz10-1 and Lan3-14 monoclonal antibodies recognized a 400 kD antigen expressed in all muscle cells. We have cloned and identified this antigen to be a member of filamin family of actin-bundling proteins. Leech Filamin consists of two calponin homology (CH) domains and 35 filamin/ABP-repeats. The CH domains bind actin while the ABP-repeats have been implicated in dimerization as well as protein-protein interactions. Using the Laz10-1 antibody, we have shown that the dorso-ventral flattener muscles develop as three discrete bundles. The middle bundle transiently expresses neuronal CAM Tractin concomitant with the formation of DP nerve. This suggests that the middle dorso-ventral muscle anlagen may provide the substrate for the axonal outgrowth and nerve formation.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Deepa V. Venkitaramani
Venkitaramani, Deepa V., "Structural and molecular analysis of leech neuronal proteins, Calsensin and Filamin " (2004). Retrospective Theses and Dissertations. 1200.