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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.


Identification of the number of bound Ca2+ ions. LC-ESI-MS data for CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F) are shown. Elution pattern of LC (top), MS of first fraction (middle), and MS of second fraction (bottom) are depicted for each sample. CCM0 (panel A) and CCMΔ4 (panel B) have only one elution peak related to 4Ca2+-bound form, and CCMΔ8 (panel F) also has only one elution peak of 3Ca2+-bound form. The molecular mass (Da) of each protein is calculated as follow: 16706.76 (CCM0), 16274.84 (CCMΔ4), 16143.65 (CCMΔ5), 16015.52 (CCMΔ6), 15916.39 (CCMΔ7), and 15769.21 (CCMΔ8).
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f2-7_35: Identification of the number of bound Ca2+ ions. LC-ESI-MS data for CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F) are shown. Elution pattern of LC (top), MS of first fraction (middle), and MS of second fraction (bottom) are depicted for each sample. CCM0 (panel A) and CCMΔ4 (panel B) have only one elution peak related to 4Ca2+-bound form, and CCMΔ8 (panel F) also has only one elution peak of 3Ca2+-bound form. The molecular mass (Da) of each protein is calculated as follow: 16706.76 (CCM0), 16274.84 (CCMΔ4), 16143.65 (CCMΔ5), 16015.52 (CCMΔ6), 15916.39 (CCMΔ7), and 15769.21 (CCMΔ8).

Mentions: We conducted LC-ESI-MS experiments to clarify the number of Ca2+ ions bound to CaM and its variants. The results are shown in Figure 2. The protein sample was passed through a reverse phase column with a mobile phase solvent containing 10 μM Ca2+. Thus, the results obtained here suggest the number of Ca2+ ions bound to CaM and its variants in the presence of 10 μM free Ca2+. The peaks appearing at different retention times were subsequently subjected to ESI-MS analyses. For all samples, the LC peaks in the ESI-MS spectra at a retention time of approximately 10.6 min show [protein+4Ca-nH](n–8)− ion. Another peak for CCMΔ5–7 appearing at a later retention time shows [variant + 2Ca-nH](n–4)− and [variant + 3Ca-nH](n–6)−. When the two peaks were observed in the LC-MS chromatogram, it was found that the former peak was related to the CaM (or variant) with a larger number of Ca2+ ions. Ca2+-binding to CaM (or variants) is tight and the on-off exchange rate is thought to be slow enough to separate the 4Ca2+-bound form from the species with smaller number of Ca2+ ions.


Roles of the C-terminal residues of calmodulin in structure and function
Identification of the number of bound Ca2+ ions. LC-ESI-MS data for CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F) are shown. Elution pattern of LC (top), MS of first fraction (middle), and MS of second fraction (bottom) are depicted for each sample. CCM0 (panel A) and CCMΔ4 (panel B) have only one elution peak related to 4Ca2+-bound form, and CCMΔ8 (panel F) also has only one elution peak of 3Ca2+-bound form. The molecular mass (Da) of each protein is calculated as follow: 16706.76 (CCM0), 16274.84 (CCMΔ4), 16143.65 (CCMΔ5), 16015.52 (CCMΔ6), 15916.39 (CCMΔ7), and 15769.21 (CCMΔ8).
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f2-7_35: Identification of the number of bound Ca2+ ions. LC-ESI-MS data for CCM0 (A), CCMΔ4 (B), CCMΔ5 (C), CCMΔ6 (D), CCMΔ7 (E), and CCMΔ8 (F) are shown. Elution pattern of LC (top), MS of first fraction (middle), and MS of second fraction (bottom) are depicted for each sample. CCM0 (panel A) and CCMΔ4 (panel B) have only one elution peak related to 4Ca2+-bound form, and CCMΔ8 (panel F) also has only one elution peak of 3Ca2+-bound form. The molecular mass (Da) of each protein is calculated as follow: 16706.76 (CCM0), 16274.84 (CCMΔ4), 16143.65 (CCMΔ5), 16015.52 (CCMΔ6), 15916.39 (CCMΔ7), and 15769.21 (CCMΔ8).
Mentions: We conducted LC-ESI-MS experiments to clarify the number of Ca2+ ions bound to CaM and its variants. The results are shown in Figure 2. The protein sample was passed through a reverse phase column with a mobile phase solvent containing 10 μM Ca2+. Thus, the results obtained here suggest the number of Ca2+ ions bound to CaM and its variants in the presence of 10 μM free Ca2+. The peaks appearing at different retention times were subsequently subjected to ESI-MS analyses. For all samples, the LC peaks in the ESI-MS spectra at a retention time of approximately 10.6 min show [protein+4Ca-nH](n–8)− ion. Another peak for CCMΔ5–7 appearing at a later retention time shows [variant + 2Ca-nH](n–4)− and [variant + 3Ca-nH](n–6)−. When the two peaks were observed in the LC-MS chromatogram, it was found that the former peak was related to the CaM (or variant) with a larger number of Ca2+ ions. Ca2+-binding to CaM (or variants) is tight and the on-off exchange rate is thought to be slow enough to separate the 4Ca2+-bound form from the species with smaller number of Ca2+ ions.

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.