Final Report Summary - LXWAP (Re-designing zinc finger proteins by swapping alpha-helical domains with foldamer helices.) Nature uses structural organisation to deliver function. For this reason, nucleic acids and proteins need to be correctly folded to display and modulate their activity. In particular, proteins use local arrangements of their peptide backbones (e.g. alpha-helices) to guarantee the correct presentation of key residues for interaction with target biomolecules. Taking inspiration from this approach, scientists created a series of artificial oligomers able to adopt helical folding (foldamers [1]), which can often be decorated with peptide-like side chains in order to mimic and eventually expand the scope of functions performed by Nature. Moving a step towards this goal, we investigated the effect of the replacement of an α-helix with an oligourea foldamer in a zinc finger protein (see Figure 1). Zinc fingers are a well described class of metalloproteins specialised in nucleic acid recognition. They are characterised by a very simple architecture, i.e. one alpha-helix and two beta-strands brought together by the coordination of a Zn atom through two cysteine and two histidine residues, and they are therefore a good model in terms of structure and function properties[2]. In particular, we selected the transcription factor Zif268, which contains three zinc fingers. In this project we investigated: 1) the synthetic feasibility of the hybrid oligourea-zinc finger domain 3 of Zif268; 2) the impact of this replacement on the folding, in particular looking at the repercussion on the metal binding ability; 3) the possibility of synthesising a whole protein containing a hybrid motif; 4) metal and DNA binding characteristic of the novel chimeric protein. The synthesis of the chimeric finger 3 of Zif268 was achieved on different scales (50-120 μmol) using a fragment condensation approach that took advantage of the presence of a glycine residue in the sequence. The C-terminal fragment was synthesised using solid phase microwave assisted methods on MBHA resin, which is compatible with the use of TFA. This allowed the use of Boc-protected succinimidyl carbamates building blocks developed in the Guichard group as precursors of oligoureas with peptide-like side chains, which were assembled adapting conditions previously developed [3]. The N-terminal fragment was synthesised on 2-chlorotrityl resin using Fmoc solid phase methods, cleaved maintaining the side protections and coupled to the C-terminal portion still on resin. As a reference, we also synthesised the native domain 3 of Zif268, using classic Fmoc microwave assisted solid phase chemistry.To have insights on the impact of the oligourea on the folding properties of the zinc finger motif we investigated its metal binding ability recurring to different techniques, i.e. UV, CD, ESI-MS and NMR. We used UV to evaluate the Kd of the Zn2+ complex by a competition experiment with Co2+. The latter displays d-d transitions in the visible spectrum when involved in a tetrahedral complex, and the disappearance of these bands upon titration of the Co2+ complex with Zn2+ is an indirect evidence of Zn binding. The Kd estimation for the Zn2+ complex was of 10-11 M, and the end point of the titration suggested the formation of a 1:1 complex. Similar properties were observed for the native peptide, although interestingly the kinetic of Zn2+ binding was much slower, but slightly thermodynamically favored (i.e. lower Kd). Using CD instead it was possible to monitor directly the effect of Zn2+ binding looking at the conformational changes in the molecule. For the native peptide the CD profile was typical for a beta-beta-alpha arrangement of the tertiary structure upon metal addition. On the contrary, the positive ellipticity observed for the chimeric motif around 200 nm is a sign of oligourea helicity. Titrations reached a plateau in both cases at 1 equivalent of Zn2+, which also supports the 1:1 stoichiometry hypothesis. The chimeric motif was also studied by 1H NMR, which showed a considerable reorganization of the system upon metal binding, The urea and amide NH signals are much more disperse in the 1H spectrum of the Zn2+ complex in comparison with the molecule alone. The N-H HSQC of the complex shows a very unique and unambiguous set of peaks, which suggests the existence of one main species in solution. To confirm the stoichiometry of the complex we also used ESI-MS, and indeed only the 1:1 complexes with Zn were observed in the state of charge 4+ and 3+ for both molecules. The correct stoichiometry and the strong Zn binding (low Kd) indicated the correct presentation of the Cys and His residues, and therefore suggested that the chimeric motif could be a good mimetic for the native one. We thus proceeded to the synthesis of the full Zif268 protein containing a chimeric finger 3. We synthesised the protein as three independent fragments (fragment 1: res. 1-36; fragment 2: res. 36-64; fragment 3: res. 65-86), using classic SPS Fmoc compatible chemistry for fragments 1 and 2, and the chemistry previously described for 3. These building blocks were after combined by ligation methods based on the chemistry of the bis(2-sulfanylethyl) amido group (SEA [4]). This technology allowed the one pot sequential assembly of the three fragments in N to C terminus direction, and the oligourea chimeric Zif268 (uZif268) was obtained in excellent purity. The formation of a 1:3 complex with Zn2+ was observed by ESI-MS, and in this context no other stoichiometries were detected. This suggested that the protein could be correctly folded. Crystallographic structural characterisation is still ongoing, as well as the assessment of the DNA binding properties of this novel hybrid biomolecule. However by ESI-MS it was already possible to observe the existence of a DNA-uZif268 complex when the composite protein was incubated in the presence of the target DNA for Zif268.In conclusion, we demonstrated that oligourea helices are good mimetics of natural alpha-helices. The chimeric zinc finger motif was successfully synthesised and retained Zn2+ binding characteristics similar to native motifs. We were able to assemble the chimeric protein uZif268 and preliminary results regarding its folding and its DNA binding properties were encouraging. When more detailed structural information will be available, we could envisage the possibility of designing zinc fingers with customised properties, having recognition features beyond their natural models. This could find application in the biological and medical fields, and could constitute a milestone towards artificial proteins.references : [1] Guichard, G.; Huc, I. Chem. Commun. (Camb) 2011, 47, 5933-41[2] Klug, A. Annu. Rev. Biochem. 2010, 79, 213–231[3] Nelli, Y.R.; Douat-Casassus, C.; Claudon, P.; Kauffmann, B.; Didierjean, C.; Guichard, G. Tetrahedron 2012, 68, 4492-4500[4] Ollivier, N.; Vicogne, J.; Vallin, A.; Drobecq, H.; Desmet, R.; El Mahdi, O.; Leclercq, B.; Goormachtigh, G.; Fafeur, V.; Melnyk, O. Angew. Chem. Int. Ed. 2012, 51, 209 –213 Related documents final1-20150605-mc-summary-fig-1.pdf
