Calcium(2+) and magnesium(2+) binding characteristics of Chlamydomonas centrin domains

摘要

Centrin is a calcium binding protein that belongs to the EF-hand superfamily, and is found in the basal bodies and the flagellar apparatus of Chlamydomonas reinhardtii. This protein is highly conserved, and has two domains. Both domains (amino-terminal and carboxyl-terminal) can be expressed and studied independently, maintaining the secondary structure as in the full-length protein. Bacterially over-expressed full-length protein and terminal domain peptides were purified and used in calcium and magnesium titration studies. These studies were performed using Fourier transform infrared (FT-IR) spectroscopy to monitor changes in the protein conformation upon addition of calcium/magnesium. Aliquots from this titration were used for quantitative determination of free calcium concentration using a calcium selective electrode. From these biophysical studies, the saturation of each terminal peptide domain and full-length protein was established, and detailed structural information at the local and overall level were determined.