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Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into [alpha] and [beta] families. Structures of several [beta]-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., [Cys.sup.1]-[Cys.sup.5], [Cys.sup.2]-[Cys.sup.4], and [Cys.sup.3]-[Cys.sup.6]. We found that human [beta]-defensin 3 (hBD3), a recently described member of the growing [beta] family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of [Cys.sup.1]-[Cys.sup.5] and of [Cys.sup.1]-[Cys.sup.6], we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native [beta] pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by [alpha]-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human [beta]-defensins and the design of novel peptide antibiotics.

Cobalamin-dependent methionine synthase catalyzes the transfer of methyl from methylcobalamin enzymes to homocystein, in E. coli, producing cob(I)alamin enzyme and methionine. AdoMet has essential to maintain methioine synthase activity. It is demonstrated that the reorganization of cob(II)alamin ligand is a rate liming step in the reductive methylation pathway. The immediate product of reductive methylation was shown to be an enzyme with a similar spectrum to the methylcobalamin His759Gly.

The interaction between flavodoxin and methionine synthase in Escherichia coli was investigated. A 1:1 complex of flavodoxin with methionine synthase showed modifications in both the visible absorbance and electron paramagnetic resonance spectra of the methionine synthase-bound cobalamin cofactor. These spectral changes are indicative of a change in coordination geometry of the cobalt in the bound cobalamin. Findings showed that E. coli flavodoxin not only provides electrons for the activation of methionine synthase but may also cause a global conformation change within methionine synthase when it binds.

To estimate the relationship between musical preference and perceived loudness, 25 subjects, musicians and nonmusicians, were asked to match the loudness of a neutral stimulus to a given musical selection. Ten different types of music were used; each was rated for likability by the subject. Over-all analysis of loudness ratings indicated that nonmusicians were more accurate in matching loudness of a neutral stimulus with the musical selections. This finding suggests differences in loudness perception between musicians and nonmusicians. Both groups were most accurate on the selection which could be inferred as most familiar.

Mammals generate a diverse array of antimicrobial proteins, largely represented by defensins or cathelicidins. The direct in vitro microbicidal activity of antimicrobial proteins has long been considered an important innate immune defense, although the in vivo relevance has only very recently been established for certain defensins and cathelicidins. Mammalian defensins and cathelicidins have also been shown to have multiple receptor-mediated effects on immune cells. Beta-defensins interact with CCR6; murine β-defensin-2 in addition activates TLR4. Cathelicidins act on FPRL1-expressing cells. Furthermore, several defensins have considerable immunoenhancing activity. Thus, it appears that mammalian antimicrobial proteins contribute to both innate and adaptive antimicrobial immunity. [ABSTRACT FROM AUTHOR]

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into α and β families. Structures of several β-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys¹-Cys[sup 5];, Cys²-Cys[sup 4], and Cys³-Cys[sup 6]. We found that human β-defensin 3 (hBD3), a recently described member of the growing β family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys¹-Cys&sup5; and of Cys¹-Cys&sup6;, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native β pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coil, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by α-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human β-defensins and the design of novel peptide antibiotics. [ABSTRACT FROM AUTHOR]

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P212121, a = 126.4, b = 41.10, c = 69.15 Å, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 Å, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 ( Anacystis nidulans). Data extending to 1.8-Å resolution were collected at 140 K and the structure was refined to an Rworkof 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 Å,) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 Å ( R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase. [ABSTRACT FROM AUTHOR]

Thymus and activation-regulated chemokine (TARC) is a CC chemokine that is most highly expressed in the thymus. TARC interacts primarily with the CCR4 receptor and to a lesser extent with the CCR8 receptor. Three different crystal forms of synthetically prepared TARC were grown in triclinic, hexagonal and tetragonal systems. The X-ray data for the triclinic crystals (unit-cell parameters a = 56.46, b = 76.48, c = 88.37 Å, α = 85.8, β = 72.8, γ = 70.0°) extend to 1.85 Å on a conventional radiation source. The hexagonal crystals diffracted to 2.2 Å at a synchrotron-radiation source and belong to either space group P6[sub 1]22 or P6[sub 5]22, with unit-cell parameters a = 61.8, c = 315. Å. The tetragonal crystals diffracted to about 5 Å at a synchrotron-radiation source and had approximate unit-cell parameters a = b = 47.7, c = 58.2 Å. [ABSTRACT FROM AUTHOR]

Human beta-defensin 3 (hBD3) is a highly basic 45-amino-acid protein that acts both as an antimicrobial agent and as a chemoattractant molecule. Although the nature of its antimicrobial activity is largely electrostatic, the importance of the molecular structure on this activity is poorly understood. Two isoforms of hBD3 were synthesized: the first with native disulfide linkages and the second with nonnative linkages. In a third synthetic peptide, all cysteine residues were replaced with alpha-aminobutyric acid, creating a completely linear peptide. A series of six small, linear peptides corresponding to regions of hBD3 with net charges ranging from +4 to +8 (at pH 7) and lengths ranging from 9 to 20 amino acids were also synthesized. The linear full-length peptide showed the highest microbicidal activity against Escherichia coli and Staphylococcus aureus, while all three full-length forms showed equal activity against Candida albicans. The linear peptide also showed high activity against Enterococcus faecium and Pseudomonas aeruginosa. Peptides corresponding to the C terminus showed higher activities when tested against E. coli, with the most active peptides being the most basic. However, only the peptide corresponding to the N terminus of hBD3 showed any activity against S. aureus and C. albicans. Further, N-terminal deletion mutants of native hBD3 showed diminished activities against S. aureus. Thus, the antimicrobial properties of hBD3 derivatives are determined by both charge and structure.