| Summary: | The work presented here probes a variety of non covalently interacting biological systems by electrospray mass spectrometry (ESIMS). Two systems were studied in depth. Cyclophilin A (CypA), an important protein in the immune system which forms a strong non covalent complex with Cyclosporin (CsA). Conditions required to preserve the complex between CypA and CsA were investigated. The optimised conditions were used to analyse a variety of CsA peptides analogues and synthetic mimics’ affinity for CypA, culminating in the screening of a combinatorial library of potential CypA inhibitors. Calmodulin (CaM) is an acidic ubiquitous calcium binding protein, which undergoes a large conformation change upon binding four Ca<sup>2+ </sup>ions. Ca<sup>2+ </sup>loaded CaM interacts with nitric oxide synthase (NOS) enzymes, which in turn control the production of the signalling molecule and cytotoxin NO. The interactions between CaM and the FMN region of nNOS are probed by ESIMS and the 46 kDa complex formed by CaM:nNOS has been retained in the gas phase. This is shown to be exclusively selective for CaM.4Ca<sup>2+</sup> Further characterisation of this system is afforded by examining a complex of CaM with a 22 residue synthetic peptide which is the proposed CaM binding site of nNOS. The strength of the interaction was examined using collision induced dissociation at a variety of charge states. Affinities of magnesium, strontium, lead, and cadmium for the calcium binding sites of CaM have also been investigated by ESIMS. All of these divalent cations behave differently with respect to CaM. Analysis of non covalent interactions by ESIMS is shown to be a powerful technique which compliments other physical structural analysis techniques and opens new avenues to study effects of toxic metal ion binding.
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