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| Home > Publications database > Kritische Aminosäurepositionen für die Bindung von ApoCalmodulin an die Stickoxidsynthas Tpy I und II |
| Home > Publications database > Kritische Aminosäurepositionen für die Bindung von ApoCalmodulin an die Stickoxidsynthas Tpy I und II |
| Dissertation / PhD Thesis/Book | PreJuSER-44612 |
2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/20240
Report No.: JUEL 3920
Abstract: Calmodulin (CaM) is a ubiquitous Ca$^{2+}$-sensorprotein, which belongs to the superfamily of EF-hand-proteins. In its Ca$^{2+}$ bound form it regulates a variety of signalling pathways and cellular processes. The Ca$^{2+}$ free form (ApoCaM) also binds to some target proteins and its physiological role has long been underestimated. The nitric oxide synthase (NOS) isoforms all have a classical Ca$^{2+}$/CaM binding motif and need CaM as a cofactor for nitric oxide synthesis. Both, NOS-I and NOS-III are activated by CaM in a Ca$^{2+}$-dependent way, whereas NOS-II binds to CaM even in absence of Ca$^{2+}$. Comparing the CaM-binding sites of NOS-I and NOS-II, four non conservative amino acid exchanges at positions 3, 6, 9, and 13 of the CaM-binding motif (NOS-I : aa 731-744; NOS-II : aa 509-522) become evident. To investigate their role in the CaM-binding of both isoforms, peptides of the CaM-binding sites with reciprocally exchanged amino acids were synthesized. Enzyme-mutants with the same exchanges were also constructed. CaM-binding studies with the peptides revealed, that the exchange of position 6 in the NOS-I CaM-binding site results in ApoCaM binding. Further exchanges at positions 3, 9, and 13 enhanced the affinity for ApoCaM. Mutations at the critical positions in the NOS-II CaM-binding site resulted in a decrease of ApoCaM affinity. The peptide with a single mutation at position 6 bound neither ApoCaM nor Ca$^{2+}$/CaM. A striking difference lay in the thermodynamic of peptide CaM-binding. The binding of NOS peptides to Ca$^{2+}$/CaM was enthalpically favoured, whereas the binding to ApoCaM was entropically driven. Classical ApoCaM-binding motifs (IQ-motifs) show a highly conserved positive charge at position 6. This positive amino acid is also found in the NOS-II CaM-binding sequence and appears to be decisive for ApoCaM binding. Decrease of the NOS-II CaM-binding site's hydrophobic character especially in the N-terminal part leads to an decrease in ApoCaM affinity. The N-terminal part of the motif seems to be responsible for ApoCaM-interaction, as it is the case for IQ-motifs. The mutations had no significant effect on the Ca$^{2+}$ dependent activation of heterologously expressed enzymes.
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