Limits...
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.


1H-NMR spectra of CaM and its variants at various concentration of Ca2+. Panels A and B are results of CCMΔ4 and CCMΔ5, respectively. The numbers shown in the two figures indicate the [Ca2+]/[variant] ratio. The assignments were according to Figure 6.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036782&req=5

f5-7_35: 1H-NMR spectra of CaM and its variants at various concentration of Ca2+. Panels A and B are results of CCMΔ4 and CCMΔ5, respectively. The numbers shown in the two figures indicate the [Ca2+]/[variant] ratio. The assignments were according to Figure 6.

Mentions: For the first NMR analysis, we demonstrated one dimensional 1H-NMR experiments to clarify the Ca2+-binding order among the four EF hands in CaM and its variants. Figure 5 indicates stacked plots of the down field amide region in one-dimensional 1H-NMR spectra of CCMΔ4 and CCMΔ5 through the Ca2+-titration.


Roles of the C-terminal residues of calmodulin in structure and function
1H-NMR spectra of CaM and its variants at various concentration of Ca2+. Panels A and B are results of CCMΔ4 and CCMΔ5, respectively. The numbers shown in the two figures indicate the [Ca2+]/[variant] ratio. The assignments were according to Figure 6.
© Copyright Policy
Related In: Results  -  Collection

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

f5-7_35: 1H-NMR spectra of CaM and its variants at various concentration of Ca2+. Panels A and B are results of CCMΔ4 and CCMΔ5, respectively. The numbers shown in the two figures indicate the [Ca2+]/[variant] ratio. The assignments were according to Figure 6.
Mentions: For the first NMR analysis, we demonstrated one dimensional 1H-NMR experiments to clarify the Ca2+-binding order among the four EF hands in CaM and its variants. Figure 5 indicates stacked plots of the down field amide region in one-dimensional 1H-NMR spectra of CCMΔ4 and CCMΔ5 through the Ca2+-titration.

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.