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Structure/calcium affinity relationships of calmodulin site III : testing the acid-pair Hypothesis using calmodulin mutants

University of British Columbia

The overall objective of this study was to test the Acid Pair Hypothesis in the calcium binding site III of calmodulin using calmodulin mutants. The Acid Pair Hypothesis was proposed to predict calcium binding affinity of a helix-loop-helix calcium binding motif based on the number and the location of acidic amino acid residues in chelating positions [Reid & Hodges, (1980) J. Theor. Biol. 84, 401-444]. This hypothesis states that a site will have a higher affinity for calcium if the anionic ligands are paired on the axial vertices of a near octahedron than if they are unpaired. The mutants were designed so that there were either three or four acidic chelating residues with acid-pairs on the X and/or Z axis. The F92W/D133E mutations were maintained in all mutants. Tryptophan was introduced as a fluorescent label into site III to monitor the calcium-induced structural transitions in the Cterminal domain. The D133E mutation in the +Z position of site IV was designed to inactivate this site with respect to calcium binding therefore eliminating the cooperative interactions between sites III and IV. The calcium affinity of site III increased when the number of the acidic chelating residues increased from three to four, when the number of acid-pairs increased from zero to one and further to two, and when the location of the acidpair was changed from the X axis to the Z axis. These results are consistent with the prediction of the Acid Pair Hypothesis. The fact that the D133E mutation drastically reduced calcium affinity of site IV indicates that the type of acidic residue in chelating positions also plays a role in dictating calcium affinity of the helix-loop-helix site. Conclusions drawn from earlier studies using synthetic models of a single helix-loop-helix calcium binding site describing the effect of the number and location of acidic residues on calcium affinity appear to be applicable to the multisite protein.

Thesis/Dissertation

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2009-04-17

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http://hdl.handle.net/2429/7305

Pharmaceutical Sciences

University of British Columbia

UBCV

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