Conclusion:Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. General significance:The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipatic, since this feature seems to help the toroidal pores formation process. Highlights A new Temporin B analogue, TB_KKG6A, was designed.TB_KKG6A is active against Gram-positive and Gram-negative bacteria.The peptide strongly interacts with the LPS of E.coli.The peptide folds into a kinked helix upon interaction with LPS. The peptide shows very low hemolytic activity.

In the funneled landscape, proteins fold to their native states through a stochastic process in which the free energy decreases spontaneously and unfolded, transition, native, and possible intermediate states correspond to local minima or saddle points. Atomic description of the folding pathway appears therefore to be essential for a deep comprehension of the folding mechanism. In metallo-proteins, characterization of the folding pathways becomes even more complex, and therefore, despite their fundamental role in critical biological processes, little is known about their folding and assembly. The study of the mechanisms through which a cofactor influences the protein folding/unfolding reaction has been the rationale of the present study aimed at contributing to the search for cofactors' general roles in protein folding reactions. In particular, we have investigated the folding pathway of two homologous proteins, Ros87, which contains a prokaryotic zinc finger domain, and Ml452-151, lacking the zinc ion. Using a combination of CD, DSC and NMR techniques, we determined the thermodynamics and the structural features, at an atomic level, of the thermal unfolding of Ros87 and compared them to the behavior of Ml452-151. Our results, also corroborated by NMR (1)H/(2)H exchange measurements, show that the presence of the structural Zn(II) in Ros87 implies a switch from the Ml452-151 fully cooperative to a two-step unfolding process in which the intermediate converts to the native state through a downhill barrierless transition. This observation, which has never been reported for any metal ion so far, may have a significant role in the understanding of the protein misfolding associated with the presence of metal ions, as observed in neurodegenerative diseases.

The exact evolutionary origin of the zinc finger (ZF) domain is unknown, as it is still not clear from which organisms it was first derived. However, the unique features of the ZF domains have made it very easy for evolution to tinker with them in a number of different manners, including their combination, variation of their number by unequal crossing-over or tandem duplication and tuning of their affinity for specific DNA sequence motifs through point substitutions. Classical Cys2His2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys2His2 ZF domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens and recently characterized. The present work focuses on the evolution of the classical ZF domains with the goal of trying to determine whether eukaryotic ZFs have evolved from the prokaryotic Ros-like proteins. Our results, based on computational and experimental data, indicate that a single insertion of three amino acids in the short loop that separates the β-sheet from the α-helix of the Ros protein is sufficient to induce a structural transition from a Ros like to an eukaryotic-ZF like structure. This observation provides evidence for a structurally plausible and parsimonious scenario of fold evolution, giving a structural basis to the hypothesis of a horizontal gene transfer (HGT) from bacteria to eukaryotes.

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