The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme–protein interactions is still missing. We report an in-depth structural analysis of Fe^III heme–peptide complexes by a combination of UV/Vis, resonance Raman, and 2D-NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine-based peptides display different heme-binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme-unbound state. Thus, the heme-binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme-regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.

The oxidation of the conotoxin μ-SIIIA in different ionic liquids was investigated, and the results were compared with those obtained in [C₂mim][OAc]. Conversion of the reduced precursor into the oxidized product was observed in the protic ILs methyl- and ethylammonium formate (MAF and EAf, respectively), whereas choline dihydrogenphosphate and Ammoeng 110 failed to yield folded peptide. However, the quality and yield of the peptide obtained in MAF and EAF were lower than in the case of the product from [C₂mim][OAc]. Reaction conditions (temperature, water content) also had an impact on peptide conversion. A closer look at the activities of μ-SIIIA versions derived from an up-scaled synthesis in [C₂mim][OAc] revealed a significant loss of the effect on ion channel Na_V1.4 relative to the buffer-oxidized peptide, whereas digestion of either μ-SIIIA product by trypsin was unaffected. This was attributed to adherence of ions from the IL to the peptide, because the disulfide connectivity is basically the same for the differentially oxidized μ-SIIIA versions.

Tridegin, a 66-mer peptide isolated from the leech Haementeria ghilianii, is a potent inhibitor of the coagulation factor XIIIa. This paper describes the chemical synthesis of tridegin by two different strategies—solid-phase assembly and native chemical ligation—both followed by oxidation in solution phase. Tridegin and truncated analogues were examined for their activity and revealed a particular importance of the C-terminal region of the parent peptide. Based on these studies a minimal sequence required for factor XIIIa inhibition could be identified. Our data revealed that the glutamine residue at position 52 (Q⁵²) of tridegin most likely binds to the active site of factor XIIIa and was therefore suggested to react with the enzyme. The function of the N-terminal region is also discussed, as the isolated C-terminal segment of tridegin lost its inhibitory activity rapidly in the presence of factor XIIIa, whereas this was not the case for the full-length inhibitor.