ABSTRACT
Bacteriocins belong to the wide variety of antimicrobial ribosomal peptides synthesised by bacteria. Enterococci are Gram-positive, catalase-negative bacteria that produce lactic acid as the major end product of glucose fermentation. Many enterococcal strains produce bacteriocins, named enterocins. We describe in this work, the structural characterisation of the 44 residues-long enterocin EJ97, produced by Enterococcus faecalis EJ97. To this end, we have used a combined theoretical and experimental approach. First, we have characterised experimentally the conformational properties of EJ97 in solution under different conditions by using a number of spectroscopic techniques, namely fluorescence, CD, FTIR and NMR. Then, we have used several bioinformatic tools as an aid to complement the experimental information about the conformational properties of EJ97. We have shown that EJ97 is monomeric in aqueous solution and that it appears to be chiefly unfolded, save some flickering helical- or turn-like structures, probably stabilised by hydrophobic clustering. Accordingly, EJ97 does not show a cooperative sigmoidal transition when heated or upon addition of GdmCl. These conformational features are essentially pH-independent, as shown by NMR assignments at pHs 5.9 and 7.0. The computational results were puzzling, since some algorithms revealed the natively unfolded character of EJ97 (FoldIndex, the mean scaled hydropathy), whereas some others suggested the presence of ordered structure in its central region (PONDR, RONN and IUPRED). A future challenge is to produce much more experimental results to aid the development of accurate software tools for predicting disorder in proteins.
Subject(s)
Bacteriocins/chemistry , Amino Acid Sequence , Bacteriocins/metabolism , Circular Dichroism , Computational Biology , Computer Simulation , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Annotation , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Denaturation , Protein Folding , Spectrometry, Fluorescence , Spectroscopy, Fourier Transform InfraredABSTRACT
The Ring1B is a core subunit protein of the PRC1 (polycomb repressive complex 1), which plays key roles in the regulation of the Homeobox gene expression, X-chromosome inactivation, stem cell self-renewal, and tumorigenesis. The C-terminal region of Ring1B interacts with RYBP, a transcriptional repressor in transiently transfected cells, and also with M33, another transcriptional repressor involved in mesoderm patterning. In this work, we show that the C-terminal domain of Ring1B, C-Ring1B, is a dimer in solution, with a dissociation constant of 200 microM, as shown by NMR, ITC, and analytical gel filtration. Each monomer is stable at physiological conditions in a wide pH range ( approximately 5 kcal mol-1 at 298 K), with a well-formed core and a spherical shape. The dimer has a high content of alpha-helix and beta-sheet, as indicated by FTIR spectra, and it is formed by the mutual docking of the preformed folded monomers. Since the C-terminal region is important for interaction with other proteins of the PRC1, the dimerization and the presence of those well-structured monomers might be a form of regulation.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Chromatography, Gel , DNA-Binding Proteins/isolation & purification , Dimerization , Hydrogen-Ion Concentration , Mice , Molecular Sequence Data , Polycomb Repressive Complex 1 , Protein Binding , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , Repressor Proteins , Ubiquitin-Protein LigasesABSTRACT
Fur (ferric uptake regulator) is a key bacterial protein that regulates iron acquisition and its storage, and modulates the expression of genes involved in the response to different environmental stresses. Although the protein is involved in several regulation mechanisms, and members of the Fur family have been identified in pathogen organisms, the stability and thermodynamic characterization of a Fur protein have not been described. In this work, the stability, thermodynamics and structure of the functional dimeric Fur A from Anabaena sp. PCC 7119 were studied by using computational methods and different biophysical techniques, namely, circular dichroism, fluorescence, Fourier-transform infrared, and nuclear magnetic resonance spectroscopies. The structure, as monitored by circular dichroism and Fourier-transform infrared, was composed of a 40% of alpha-helix. Chemical-denaturation experiments indicated that Fur A folded via a two-state mechanism, but its conformational stability was small with a value of DeltaG = 5.3 +/- 0.3 kcal mol(-1) at 298 K. Conversely, Fur A was thermally a highly stable protein. The high melting temperature (Tm = 352 +/- 5 K), despite its moderate conformational stability, can be ascribed to its low heat capacity change upon unfolding, DeltaCp, which had a value of 0.8 +/- 0.1 kcal mol(-1) K(-1). This small value is probably due to burial of polar residues in the Fur A structure. This feature can be used for the design of mutants of Fur A with impaired DNA-binding properties.
