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

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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.

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Ca2+-binding by CaM and its variants. The flow dialysis results for CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8 are shown. The experimental conditions have been described in detail in Materials and Methods.
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f3-7_35: Ca2+-binding by CaM and its variants. The flow dialysis results for CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8 are shown. The experimental conditions have been described in detail in Materials and Methods.

Mentions: Next, we measured the Ca2+-affinity of CaM and the variants. The results of the flow dialysis experiments with CaM and a series of variants are shown in Figure 3. The results indicate that all of the variants are less sensitive than CCM0 to Ca2+; all these variants bind at least three Ca2+ ions. Although very high Ca2+ concentrations may have a tendency to lead to great ambiguity in the flow dialysis data, unclear results could be caused by the truncated EF4. Interestingly, EF3 can bind Ca2+ in all variants, although it is coupled with EF4 to form the C-lobe.


Roles of the C-terminal residues of calmodulin in structure and function
Ca2+-binding by CaM and its variants. The flow dialysis results for CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8 are shown. The experimental conditions have been described in detail in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036782&req=5

f3-7_35: Ca2+-binding by CaM and its variants. The flow dialysis results for CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8 are shown. The experimental conditions have been described in detail in Materials and Methods.
Mentions: Next, we measured the Ca2+-affinity of CaM and the variants. The results of the flow dialysis experiments with CaM and a series of variants are shown in Figure 3. The results indicate that all of the variants are less sensitive than CCM0 to Ca2+; all these variants bind at least three Ca2+ ions. Although very high Ca2+ concentrations may have a tendency to lead to great ambiguity in the flow dialysis data, unclear results could be caused by the truncated EF4. Interestingly, EF3 can bind Ca2+ in all variants, although it is coupled with EF4 to form the C-lobe.

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.


Related in: MedlinePlus