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Target recognition by calmodulin

Skinner, MA; (1999) Target recognition by calmodulin. Doctoral thesis, University of London. Green open access

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Abstract

Calmodulin (CaM) inhibits mammalian phosphofructokinase (PFK) in a calcium dependent manner; however the putative CaM binding sequence is not homologous to those of other well characterized systems. This work addresses the structural and functional properties of complexes of CaM with selected target peptides from PFK using calcium binding and optical spectroscopic methods. Wild-type CaM, site-directed mutants of CaM, and cleaved domains of CaM were studied in complex with PFK target peptides of different length and amino-acid composition. Comparison of equilibria and structural data from these complexes allowed definition of the sequence involved in the interaction and conformation of the complexes. The effect of binding different peptides on the calcium affinity of CaM has been determined by an indicator method. The presence of the PFK target peptide apparently causes a 200 fold increase in the affinity of the first two stoichiometric binding constants (K1 & K2), however there is little effect on K3 & K4. Interestingly the change in far-UV, near-UV CD. and fluorescence spectroscopic signals generated by the interaction of CaM with the target sequences are essentially complete on addition of two molar equivalents of calcium to 1 molar equivalent of CaM:PFK peptide complex, consistent with a differential function of the two domains. The dissociation rates of calcium from CaM:PFK peptide complexes has been determined in stopped-flow experiments using the calcium chelators EGTA and Quin-2. Calcium dissociation kinetics are biphasic, with 2 sites having higher affinity. Enzyme activity studies show PFK inactivation to be CaM concentration dependent and the addition of PFK target peptide reverses the inhibition. A mechanism for the calcium dependent interaction of CaM with the PFK target sequence is presented. In comparison to the three closely related known CaM:target peptide structures the interaction of CaM with the PFK target sequence apparently results in a complex with a conformation of lower peptide helicity and with significantly less calcium dependence of interactions. Hence the CaM:PFK complex appears to define a new type of CaM interaction, distinctively different in structure from that currently considered for the binding of CaM in apo- or holo-form to sequences from other target enzymes. This novel interaction has also been addressed by crystallographic methods and details of optimal crystallization conditions for the CaM:PFK peptide complex are described.

Type: Thesis (Doctoral)
Title: Target recognition by calmodulin
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
UCL classification: UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of)
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1514509
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