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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/analyst Page 1 of 24 Analyst The use of Ion Mobility Mass Spectrometry to assist Protein Design: a Case 1 2 3 Study on Zinc Finger Fold versus Coiled Coil Interactions 4 5 Yana Berezovskaya b, Massimiliano Porrini b,1, Chris Nortcliffe a, and Perdita E Barran*a 6 7 a The School of Chemistry, Manchester Institute of Biotechnology, 8 The University of Manchester, Manchester, UK 9 b 10 School of Chemistry, University of Edinburgh, Edinburgh, UK 11 12 * Corresponding author ([email protected]) 13 14 15 Abstract 16 17 18 The dramatic conformational change in zinc fingers on binding metal ions for DNA 19 20 recognition makes their structure-function behaviour an attractive target to mimic in de novo 21 22 designed peptides. Mass spectrometry, with its high throughput and low sample consumption 23 24 provides insight into how primary amino acid sequence can encode stable tertiary fold. We 25 26 present here the use of ion mobility mass spectrometry (IM-MS) coupled with molecular 27 Manuscript 28 29 dynamics (MD) simulations as a rapid analytical platform to inform de novo design efforts for 30 31 peptide-metal and peptide-peptide interactions. A dual peptide-based synthetic system, ZiCop 32 33 based on a zinc finger peptide motif, and a coiled coil partner peptide Pp, have been 34 35 investigated. Titration mass spectrometry determines the relative binding affinities of different 36 37 Accepted divalent metal ions 1 as Zn 2+ > Co 2+ >> Ca 2+ . With collision induced dissociation (CID), we 38 39 40 probe complex stability, and establish that peptide-metal interactions are stronger and more 41 42 ‘specific’ than those of peptide-peptide complexes, and the anticipated hetero-dimeric complex 43 44 is more stable than the two homo-dimers. Collision cross-sections (CCS) measurements by IM- 45 Analyst 46 MS reveal increased stability with respect to unfolding of the metal-bound peptide over its apo- 47 48 49 form, and further, larger collision cross sections for the hetero-dimeric forms suggest that 50 51 52 53 54 1 Current address :- Institut Européen de Chimie et Biologie (IECB) 55 CNRS UMR 5248 Chimie et Biologie des Membranes et des Nano-objets (CBMN) 56 2, rue Robert Escarpit 57 33607 Pessac Cedex 58 FRANCE 59 60 1 Analyst Page 2 of 24 dimeric species formed in the absence of metal are coiled coil like. MD supports these 1 2 structural assignments, backed up by data from visible light absorbance measurements. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Manuscript 28 29 30 31 32 33 34 35 36 37 Accepted 38 39 40 41 42 43 44 45 Analyst 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Page 3 of 24 Analyst Introduction 1 2 One of the important goals of protein design is to replicate, from first principles, active 3 4 2 5 folds, and on doing this synthesise small functional candidates, that may perform useful tasks . 6 7 The usual approach involves careful consideration of which active fold(s) are required, 8 9 identification of the minimal sequence features to retain fold, followed by iterative design 3. 10 11 Assessing whether a given design strategy has been successful in obtaining the correct fold, 12 13 will usually be achieved with several analytical biophysical characterisation methods, with 14 15 16 mass spectrometry usually only employed to obtain the weight (and perhaps purity) of the 17 4 18 synthesised component(s). However developments in soft ionisation techniques , and in 19 20 particular in electrospray ionisation 5 have enabled researchers to analyse intact protein 21 22 complexes in the gas phase, determine stoichiometries and detail interactions of systems of 23 24 6 25 increasing complexity . Collision induced dissociation can be applied to interrogate the 26 7, 8 27 strength of interaction between protein complex subunits . It has also become possible to Manuscript 28 29 determine conformations of proteins and their complexes using a related technique termed ion 30 31 mobility mass spectrometry (IM-MS) 9-11 . These mass spectrometry based approaches to 32 33 determining structure represent an attractive rapid route to analysis that might form part of a 34 35 36 protein design platform. 37 Accepted 38 In this work we present an analytical workflow that can be used to characterise and test 39 40 de novo designed peptides. As an example system we take a peptide that attempts to encode 41 42 two different sequences for two distinct folds simultaneously; the transition between the 43 44 45 conformational states is reversible and is triggered by specific metal binding. Our Analyst 46 47 characterisation platform has been developed to interrogate stoichiometry of binding, metal 48 49 affinity, fold, conformational transitions, and the strength of interactions between hetero- 50 51 dimers. We use mass spectrometry (MS) ion mobility mass spectrometry (IM-MS), molecular 52 53 dynamics and solution based measurements. This workflow is illustrated in Figure 1 along with 54 55 56 examples which appear later in this paper. 57 58 59 60 3 Analyst Page 4 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Manuscript 28 29 30 31 Figure 1. Design concept of the dual reversible peptide-based switch. A – Sequence of the ZiCop and its binding 32 partner Pp in relation to the structural requirements for the zinc finger and coiled coil designs. The ‘X’ refers to 33 34 any amino acid, ‘h’ – to any hydrophobic residue, ‘p’ – to any polar residue. Below is proposed switching 35 process: the red peptide represents ZiCop, which is responsive to zinc; the partner peptide (blue) was designed to 36 interact with ZiCop in the absence of zinc. B through D – A schematic representing analytical workflow of the 37 Accepted 38 design concept interrogation using MS platform. B – Stoichiometry of binding (top spectrum: free ZiCop peak, 39 bottom: Zn-bound peptide). C – Quantification of affinity of the peptide to the metal ion (mass spectra of Zn 40 titrations into ZiCop and resulting biding curve to yield dissociation constant). D – Evaluation of the hetero- 41 42 dimeric coiled coil association using collisionally induced dissociation (CID); resulting curves indicate survival 43 of the precursor ion of the coiled coil (purple) and formation of the products – ZiCop and Pp (red and blue 44 45 respectively). Analyst 46 47 48 The two conformations of the test switching system are a zinc finger fold and a coiled- 49 50 51 coil motif. The choice has been dictated by the fact that the rules governing the folds are 52 53 extensively studied, so the novel bio-orthogonal systems can be designed confidently. Also, 54 55 both folds are independent, stable and well-defined motifs. The reversible switch between the 56 57 two conformational states is controlled by the binding of a metal ion, zinc, to the peptide. The 58 59 60 4 Page 5 of 24 Analyst aim of synthesis was that the peptide, named ZiCop (reflecting the structural duality of zinc 1 2 finger and coiled-coil peptide), in the absence of the Zn 2+ ions would form and amphipathic 3 4 5 parallel dimeric helix for coiled-coil interactions with a partner peptide (Pp), then with the 6 2+ 7 addition of Zn form a metal-bound monomer adopting a zinc finger conformation. The 8 9 system can be switched back to the coiled-coil interactions through the removal of Zn 2+ using 10 11 EDTA. 1 12 13 The zinc finger proteins were amongst the earliest reported gene-specific eukaryotic 14 15 12 16 transcription factors . They possess a repeating motif which is involved with binding of 17 18 DNA, and also associated with zinc binding, which led to the name ‘zinc finger’. Misfolding of 19 20 zinc finger proteins has been linked to a variety of human diseases such as cancer and a series 21 22 of neural disorders 13 , highlighting a need to study the structural stability of this motif. There 23 24 14 25 are several different classes of zinc finger fold each with a different structural motif although 26 27 all involve cysteine and histidine residues binding the zinc ion. A hydrophobic cluster is also Manuscript 28 29 conserved within most zinc fingers. The zinc ion is a vital component in stabilising the 30 31 structure of these proteins: in its absence the protein has a different structural conformation. 32 33 The first discovered and most common zinc finger motif is Cys His The domains adopt a ββα 34 2 2.

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