Cysteinyl peptides likely played an important role in the prebiotic synthesis of cofactors, such as iron-sulfur clusters. However, cysteinyl peptides must be reduced in order to coordinate iron-sulfur clusters. Mixtures of ferric ions and cysteinyl peptides leads to the reduction of ferric to ferrous ions and the concomitant formation of disulfide bridged, oxidized cysteinyl peptides that are incapable of coordinating an iron-sulfur cluster. Here, we develop a photochemically driven solution to this problem. Lipoic acid (( R )-5-(1,2-dithiolane-3-yl)pentanoic acid), a molecule structurally similar to fatty acids, can be photochemically reduced and can subsequently reduce the oxidized cysteinyl peptides needed for the coordination of an iron-sulfur cluster. Other dithilane ring containing molecules possess similar activity to lipoic acid. The synthesis of small peptides containing cysteines, such as glutathione and GCG (Gly-Cys-Gly) is easy by both solid phase and solution phase methodologies. However, as the length of the peptide increases, the yield begins to decrease, especially for peptides containing cysteines due to oxidation. One solution could be to exploit a previously uncovered mechanism for the joining of peptides into longer peptides. Such mechanisms, referred to as CPL for catalytic peptide ligation, rely on either thiols or metals as catalysts and peptide nitriles as substrates. Thus far, CPL has only been exploited with non-cysteinyl peptides. In this thesis, we extend CPL to cysteine containing peptides by taking advantage of the templating effects of Zn2+. Longer peptides with properly spaced cysteines are frequently better able to stabilize iron-sulfur clusters in aqueous solution than shorter peptides. Coordination can either be complete or an open coordination position, filled by solvent, can be used to bind substrate. Two well-known examples of such an arrangement are the radical SAM (S-adenosylmethionine) enzyme and aconitase being an enzyme of the citric acid cycle. We designed and synthesized peptide sequences that could coordinate a [4Fe-4S]2+ cluster with three cysteinyl ligands, leaving an open coordination position. The stability of the [4Fe-4S] cluster was affected by the intermediates of the citric acid cycle. The iron-sulfur can be reconstituted with the long peptidyl sequences from proteins such as SLC25A39 which contains four cysteine ligands to form [2Fe-2S] cluster, which is necessary for glutathione transport from cytosol to mitochondria.

Metabolism is a subset of chemistry that allows cells to defy thermodynamic equilibrium, a fundamental process that must have been in place from the very beginning of biology. Before evolution produced efficient catalysts in the form of complex protein machinery, short metal binding peptides might have preceded modern metalloproteins. Such prebiotic, metal-binding motifs have been hypothesized to have existed through analyses of extant protein sequences. However, it is unclear how metal-binding motifs might have evolved in the harsh prebiotic environment. Here, we show how certain environments, in particular seawater-like environments rich in divalent cations and especially Mg2+, support the survival of short peptides upon extreme temperatures as high as 150 °C. Moreover, while Mg2+ does not offer the same protection from UV light, peptides are protected from both heat and irradiation when bound to a metal ion. The results suggest that specific environments rich in metal ions may be better suited for the emergence of complex systems in the path toward life. Additionally, the conditional degradation of peptides depending on their ability of binding metals might have enabled a selection mechanism that would favor the survival of metal-binding motifs which resemble the motifs found in modern proteins. These short sequences could have acted as early, simple catalysts able to facilitate a restricted set of chemical reactions, which would shape the emergence and biology of the Last Universal Common Ancestor.

Intracellular chloride and pH are fundamental regulators of neuronal excitability and they are often co-modulated during excitation-inhibition activity. The study of their homeostasis requires simultaneous measurements in vivo in multiple neurons. Combining random mutagenesis screening, protein engineering and two-photon-imaging this thesis work led to the discovery of new chloride-sensitive GFP mutants and to the establishment of ratiometric imaging procedures for the quantitative combined imaging of intraneuronal pH and chloride. These achievements have been demonstrated in vivo in the mouse cortex, in real-time monitoring the dynamic changes of ions concentrations during epileptic-like discharges, and in glioblastoma primary cells, measuring osmotic swelling responses to various drugs treatment.