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Studies of the Interaction of Calmodulin With Fluorescent Labelled Target Peptides

Kamran-Pirzaman, Shiva; (1999) Studies of the Interaction of Calmodulin With Fluorescent Labelled Target Peptides. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is < 10 -6 M and during its activation itrises to > 10 -6 M. Calmodulin (CaM) is a highly conserved, ubiquitous secondarymessenger protein that has to translate this rise in intracellular calcium into a physiological response in all eukaryotic cells. Upon binding 4 calcium ions, this small acidic protein (148 amino acids) interacts with high affinity and specificity with a multiplicity of structurally and functionally diverse enzymes such as myosin light chain kinase (skMLCK), CaM kinase II, NO synthase, etc. No single consensus sequence for recognition by CaM exists, yet the complexes formed are highly specific and of high affinity. Solved NMR and crystal structures of CaM in complex with peptides derived from several enzymes have revealed that both domains of Ca4CaM interact with portions of an essentially alpha helical target sequence. This basic model does not however account for the unique diversity of the target sequences recognised by CaM, which appears to reside in specific structural details of the interaction. Investigation of this question requires a detailed description of the processes of calcium dependent target recognition. The aim of this project has been to explore the use of fluorescent labelling as a reporter for investigating the interaction with CaM of the CBP synthetic target peptides and peptides derived from the CaM-binding sequence of skMLCK. Fluorescence spectroscopy and circular dichroism are the main techniques used to characterise the interaction of fluorescent labelled peptides with CaM, and to address the existence of possible Ca2.CaM-peptide intermediate species by spectroscopic observation. A second part of this project, based on the results obtained from the above experiments has been the design of genetically engineered, cysteine containing hybrid CaM-peptide molecules as potential optical biosensors for calcium ions for in vivo applications. The characterisation of fluorescent labelled hybrids in terms of spectroscopic properties, Ca2+ binding affinity and dissociation kinetics has been thoroughly described. In vitro calibration experiments have shown that these molecules are useful as Ca2+ indicators in the of physiological Ca2+ concentrations. In vivo experiments on different cell types have revealed an even distribution of the indicator and a fluorescence response to changes in intracellular free [Ca2+]. Comparisons of the properties of these hybrid-based indicators with the conventional Ca2+ biosensors are drawn and discussed throughout the work.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Studies of the Interaction of Calmodulin With Fluorescent Labelled Target Peptides
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10121000
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