An N-terminal half fragment of the histidine phosphocarrier protein, HPr, is disordered but binds to HPr partners and shows antibacterial properties.

BACKGROUND: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes. METHODS: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR). RESULTS: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules. CONCLUSIONS: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity. GENERAL SIGNIFICANCE: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones.

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: a biophysical characterization.

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-beta-amino acids. This enzyme is an attractive candidate for commercial production of D-aminoacids, which are used in the production of semi-synthetic beta-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH approximately 6.0, which is stable up to pH approximately 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.

Into the lipid realm: stability and thermodynamics of membrane proteins.

The first comprehensive studies on the structure and thermodynamics of membrane proteins have started revealing the exact architecture of these macromolecules and the physical-chemical rules behind their structures. In this review, the stabilities of several families of membrane proteins, obtained by using spectroscopic, calorimetric and single molecule techniques are surveyed. The data on the stability of membrane proteins are compared with those obtained in soluble proteins. The comparison indicates that although the number of particular atomic interactions is larger in membrane proteins than in soluble ones, the overall values are similar. The consensus is that some intrinsic properties of membrane proteins resemble those of soluble ones, but there are critical differences arising form the inter-molecular contacts with the surrounding membrane. Taken together, all these efforts improve our understanding of the universal forces governing protein folding, and will help in the design of membrane proteins in the near future.

Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I.

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr(9-30), spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first alpha-helix of HPr of Streptomyces coelicolor, HPr(sc). By using fluorescence and circular dichroism, we first determined qualitatively that HPr(sc) and HPr(9-30) did bind to EI(sc), the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr(9-30) and HPr(sc) for EI(sc) by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr(9-30) was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI(sc) and HPr(sc) is enthalpy driven and in other species is entropy driven; further, the affinity of HPr(sc) for EI(sc) was smaller than in other species. However, the affinity of HPr(9-30) for EI(sc) was only moderately lower than that of EI(sc) for HPr(sc), suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.

The isolated C-terminal domain of Ring1B is a dimer made of stable, well-structured monomers.

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.

The helical structure propensity in the first helix of the histidine phosphocarrier protein of Streptomyces coelicolor.

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), formed by a cascade of several proteins, mediates the uptake and phosphorylation of carbohydrates, and it is also involved in signal transduction. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or proteins fragments able to interfere the first step of the protein cascade: the phosphorylation of the HPr protein by the enzyme EI. We designed a peptide comprising the active site and the first alpha-helix of HPr of S. coelicolor; we also obtained a fragment of HPr by protein engineering methods, comprising the first forty-eight residues and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure ( approximately 7 %), and an identical free energy of helix formation. In 40 % TFE, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2D-NMR assignments of the shorter peptide, and the presence of similar NOE contacts in both fragments. These findings, with the kinetic results in other members of the HPr family, highlight the importance of short- and long-range interactions during the folding reaction of HPr proteins. Based on the residual helical population, hypothesis about the inhibition capacity of the PTS by both fragments are discussed.

Isolation and characterization of a thermostable beta-xylosidase in the thermophilic bacterium Geobacillus pallidus.

The isolation, purification, biochemical and biophysical characterization of the first reported beta-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 degrees C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 degrees C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of alpha-helix (44%) and beta-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of beta-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.

Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system.

The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg2+. The conformational stability and structure of the EI from Streptomyces coelicolor, EI(sc), were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI(sc) was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI(sc) had a well-folded structure; however, at low pH, EI(sc) showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI(sc), in the absence of PEP and Mg2+, was maximal at pH 7. The presence of PEP and Mg2+ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI(sc) was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.

Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization.

The capsid protein, CA, of HIV-1 forms a capsid that surrounds the viral genome. However, recent studies have shown that an important proportion of the CA molecule does not form part of this capsid, and its location and function are still unknown. In the present work we show, by using fluorescence, differential scanning calorimetry and Fourier-transform infrared spectroscopy, that the C-terminal region of CA, CA-C, is able to bind lipid vesicles in vitro in a peripheral fashion. CA-C had a greater affinity for negatively charged lipids (phosphatidic acid and phosphatidylserine) than for zwitterionic lipids [PC/Cho/SM (equimolar mixture of phosphatidylcholine, cholesterol and sphingomyelin) and phosphatidylcholine]. The interaction of CA-C with lipid membranes was supported by theoretical studies, which predicted that different regions, occurring close in the three-dimensional CA-C structure, were responsible for the binding. These results show the flexibility of CA-C to undergo conformational rearrangements in the presence of different binding partners. We hypothesize that the CA molecules that do not form part of the mature capsid might be involved in lipid-binding interactions in the inner leaflet of the virion envelope.

The conformational stability and thermodynamics of Fur A (ferric uptake regulator) from Anabaena sp. PCC 7119.

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.

Structure and conformational stability of the enzyme I of Streptomyces coelicolor explored by FTIR and circular dichroism.

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS), formed by a cascade of several proteins, couples the translocation and phosphorylation of specific sugars across cell membranes. The structure and thermal stability of the first protein (enzyme I, EI) of the PTS in Streptomyces coelicolor is studied by using far-UV circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) at pH 7.0. The deconvolution of FTIR spectra indicates that the protein is mainly composed by a 35% of alpha-helical structure and 30% of beta-sheet. The thermal denaturation curves, as followed by both techniques, show only a midpoint at 330 K. This thermal denaturation behaviour is different to that observed in other members of the EI family.