Preparation and characterization of a single-chain calcineurin–calmodulin complex

✍ Scribed by Yunlong Qin; Jing Liu; Xin Li; Qun Wei

• Publisher: Elsevier Science
• Year: 2005
• Tongue: English
• Weight: 526 KB
• Volume: 1747
• Category: Article
• ISSN: 1570-9639
• DOI: 10.1016/j.bbapap.2004.11.002

✦ Synopsis

Calcineurin (CN), a Ca 2+ /calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca 2+ -regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn 2+ , Ni 2+ and Mg 2+ . Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca 2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca 2+ .