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Roles of the C-terminal residues of calmodulin in structure and function

View Article: PubMed Central - PubMed

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.

No MeSH data available.


CD spectra of CaM and its variants. The green and red curves indicate the CD data with and without Ca2+, respectively. CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F).
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f4-7_35: CD spectra of CaM and its variants. The green and red curves indicate the CD data with and without Ca2+, respectively. CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F).

Mentions: The global conformation and Ca2+-induced conformational changes in CaM and its variants were monitored using CD. The CD spectra of CaM and its deletion mutants in the absence and presence of Ca2+ are shown in Figure 4. The spectra of apo and Ca2+-bound CCM0 were greatly different. Ca2+-induced conformational change caused this spectral difference for CCM0. Although CCMΔ4 displayed a large spectral change similar to that observed for CCM0, a not so large spectral change was detected for CCMΔ5. Therefore, we can point out that deletion of M144 causes this drastic difference. Like CCMΔ5, the shorter mutants lacking 6, 7, and 8 residues at the C-terminus did not exhibit significant spectral change induced by Ca2+-binding. This implies that further truncation of the C-terminal region increases insensitivity to Ca2+ and relates to reduced Ca2+-induced structural changes.


Roles of the C-terminal residues of calmodulin in structure and function
CD spectra of CaM and its variants. The green and red curves indicate the CD data with and without Ca2+, respectively. CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036782&req=5

f4-7_35: CD spectra of CaM and its variants. The green and red curves indicate the CD data with and without Ca2+, respectively. CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F).
Mentions: The global conformation and Ca2+-induced conformational changes in CaM and its variants were monitored using CD. The CD spectra of CaM and its deletion mutants in the absence and presence of Ca2+ are shown in Figure 4. The spectra of apo and Ca2+-bound CCM0 were greatly different. Ca2+-induced conformational change caused this spectral difference for CCM0. Although CCMΔ4 displayed a large spectral change similar to that observed for CCM0, a not so large spectral change was detected for CCMΔ5. Therefore, we can point out that deletion of M144 causes this drastic difference. Like CCMΔ5, the shorter mutants lacking 6, 7, and 8 residues at the C-terminus did not exhibit significant spectral change induced by Ca2+-binding. This implies that further truncation of the C-terminal region increases insensitivity to Ca2+ and relates to reduced Ca2+-induced structural changes.

View Article: PubMed Central - PubMed

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.

No MeSH data available.