Neurocalcin ẟ Response to Calcium: Which EF Hand Matters the Most?

Author(s)

Trokon Fiawoo

Date Approved

5-2016

Document Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

Department

Cell Biology

College

Graduate School of Biomedical Sciences

First Advisor

Venkataswar Venkataraman, PhD

Committee Member 1

Robert Nagele, PhD

Committee Member 2

John Pastorino, PhD

Subject(s)

Neuronal Calcium-Sensor Proteins; Neurocalcin; EF Hand Motifs; Calcium-Binding Proteins; Neurons

Disciplines

Cell Biology | Medical Cell Biology | Medical Neurobiology | Medicine and Health Sciences | Molecular and Cellular Neuroscience

Abstract

Changes in intracellular calcium levels play a very important role in cell signaling, in turn, affecting neuronal functions such as memory, learning and cell death. A class of proteins called Neuronal Calcium Sensor (NCS) proteins serves to modulate the functioning of the neuronal cells in response to changes in calcium levels. Structurally, all NCS proteins have 4 calcium-binding EF hand motifs, although EF I does not bind to calcium in many members. All NCS proteins are myristoylated at the N-terminus. Neurocalcin delta (NCALD) is an NCS protein, and shares 95% similarity in primary amino acid sequence with another major NCS protein, Hippocalcin (HPCA). Studies with NCALD have indicated that the three functional EF hands have differing affinities for calcium. Point mutations that knock out binding at each EF hand individually appear to indicate specific effects. Similar observations have been made with other NCS proteins such GCAPs. The general consensus is that the EF3 hand is the highest affinity binding site for calcium. No such information is available with HPCA. Furthermore, the relative contribution of the three EF hands to calcium-induced responses in NCALD have not been delineated. As a first step in achieving this goal, NCALD mutants were generated where binding of calcium to each EF hand was individually abolished. Three different parameters were then used to compare the proteins' response to calcium: (i) tryptophan fluorescence to monitor local changes in structure around the tryptophan residues, (ii) native gel electrophoresis to monitor global changes; and (iii) gel filtration chromatography to monitor the oligomerization state. The results challenge the current theory and suggest a more intricate relationship among the three EF hands.

Recommended Citation

Fiawoo, Trokon, "Neurocalcin ẟ Response to Calcium: Which EF Hand Matters the Most?" (2016). Graduate School of Biomedical Sciences Theses and Dissertations. 71.
https://rdw.rowan.edu/gsbs_etd/71