Search

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the -30 amino acid eukaryotic zinc finger motif in which a beta-sheet and an or-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a beta beta beta alpha alpha topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion.The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (MI4 and MI5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques.We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.(c) 2013 Published by Elsevier B.V.

Background Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. Methods Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB-G6A sequence, which was further optimized by increasing the peptide positive charge (TB-KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. Results TB-KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. 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, a 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 amphipathic, since this feature seems to help the toroidal pore formation process. © 2013 Elsevier B.V. Background Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. Methods Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB-G6A sequence, which was further optimized by increasing the peptide positive charge (TB-KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. Results TB-KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. 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, a 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 amphipathic, since this feature seems to help the toroidal pore formation process. © 2013 Elsevier B.V.

International audience; 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 Cys 2 His 2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys 2 His 2 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 b-sheet from the a-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.

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 Ml4 52-151, 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 1H/2H 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. © 2013 American Chemical Society.