Investigation of protein‐ion interactions by mass spectrometry and ion mobility mass spectrometry
Protein‐ion interactions play an important role in biological systems. A considerable number of elements (estimated 25 – 30) are essential in higher life forms such as animals and humans, where they are integral part of enzymes involved in plethora of cellular processes. It is difficult to overestimate the importance of thorough understanding of how protein‐ion interplay affects living cell in order to be able to address therapeutic challenges facing humanity. Presented to the reader’s attention is a gas‐phase biophysical analysis of peptides’ and proteins’ interactions with biologically relevant ions (Zn2+ and I–). This investigation provides an insight into conformational changes of peptides and proteins triggered by ions. Mass spectrometry and ion mobility mass spectrometry are used in this work to probe peptide and protein affinities for a range of ions, along with conformational changes that take place as a result of binding. Observation of peptide and protein behaviour in the gas phase can inform the investigator about their behaviour in solution prior to ionisation and transfer from the former into the latter phase. Wherever relevant, the gas‐phase studies are complemented by molecular dynamics simulations and the results are compared to solution phase findings (spectroscopy). Two case studies of protein‐ion interactions are presented in this thesis. Firstly, sequence‐to‐structure relationships in proteins are considered via protein design approach using two synthetic peptide‐based systems. The first system is a synthetic consensus zinc finger sequence (vCP1) that is responsive to zinc: it adopts a zinc finger fold in the presence of Zn2+ by coordinating the metal ion by two cysteines and two histidines. This peptide has been selected as a reference for the zinc‐bound state and a simple model to refine the characterisation method in preparation for analysis of a more sophisticated second system – dual conformational switch. This second system (ZiCop) is designed to adopt either of the two conformations in response to a stimulus: zinc finger or coiled coil. The reversible switch between the two conformational states is controlled by the binding of zinc ion to the peptide. Interactions of both peptide systems with a number of other divalent metal cations (Co2+, Ca2+ and Cu2+) are considered also, and the differences in binding and switching behaviour are discussed. Secondly, protein‐salt interactions are investigated using three proteins (lysozyme, cytochrome c and BPTI) using variable temperature ion mobility mass spectrometry. Ion mobility measurements were carried out on these proteins with helium as the buffer gas at three different drift cell temperatures – ‘ambient’ (300 K), ‘cold’ (260 K) and ‘hot’ (360 K), and their conformational preferences in response to HI binding and temperature are discussed.