期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1760, 期 5, 页码 745-753出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2006.02.003
关键词
calreticulin; metal ion; rotary shadowing electron microscopy; circular dichroism; calnexin; protein stability
资金
- NIAID NIH HHS [AI45070] Funding Source: Medline
Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1-180 fused by a linker to 290-400) and P-domain (189-280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region. (c) 2006 Elsevier B.V. All rights reserved.
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