Metal-binding ability of histidine-containing peptidehydroxamic acids: Imidazole versus hydroxamate coordination

Three new peptidehydroxamic acids (l-alanyl-l-histidinehydroxamic acid, l-Ala-l-HisNHOH, l-alanyl-l-alanyl-l-histidinehydroxamic acid, l-Ala-l-Ala-l-HisNHOH and l-histidyl-l-alaninehydroxamic acid, l-His-l-AlaNHOH) were synthesized and their complexation with Cu(II), Ni(II) and Zn(II) were studied by pH-potentiometric, UV-Vis, CD, ¹H NMR, EPR and ESI-MS methods. Each of the studied peptide derivatives involves one side-chain imidazole unit and the effect of this group on the metal binding of the hydroxamic moiety is evaluated in the paper. The obtained results are compared to those of the complexes of some histidine-containing di- or tripeptides and also to those of hydroxamic derivatives of aliphatic peptides.A competition between the hydroxamate and imidazole functions occurs in all systems, but the extent differs from metal to metal, from ligand to ligand and depends very much on the pH. The imidazole was found to play the most determinant role in the Cu(II) complexes, somewhat less in the Ni(II)-containing ones, while (except the case of l-Ala-l-HisNHOH) negligible role was found in the Zn(II)-complexes. Common feature of the Ni(II)- and especially Cu(II)-containing systems is that if an imidazole-N is displaced by a hydroxamate, imidazole-bridged di- and polynuclear complexes are formed.     

A new two-dimensional thallium(I) coordination polymer with 4-hydroxybenzylidene-4-aminobenzoate: Thermal, structural, solution and solvatochromic studies

A new one-dimensional thallium(I) coordination polymer with a Schiff base ligand, {[Tl(L)(HL)](H₂O)_0.77}_n (1) [HL = 4-hydroxybenzylidene-4-aminobenzoic acid], has been synthesized and characterized. The single-crystal X-ray data of compound 1 show the coordination number of the Tl^I ions to be seven. The thermal stability of 1 was studied by thermal gravimetric (TG) and differential thermal analyses (DTA). The overall stoichiometry in the solid state is 1:2, but both spectrophotometric and conductometric methods did not support formation of a complex with this stoichiometry in solution. The solvatochromic behaviour of 4-hydroxybenzylidene-4-aminobenzoic acid was also investigated by studying its spectra in a selection of different organic solvents. The observed bands are assigned to electronic transitions.     

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI_5–24). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His₆ tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.     

A disulfide bridge network within the soluble periplasmic domain determines structure and function of the outer membrane protein RCSF

RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.     

Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.     

Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.     

Microbially enhanced oil recovery from miniature model columns through stimulation of indigenous microflora with nitrate

Sandpack columns filled with heavy oil and water, with or without a nitrate-reducing inoculum, were used to determine if nitrate injections could lead to enhanced oil recovery. Production of oil and water into vials filled with helium or argon was monitored during repeated anaerobic incubations at 30 °C. Regardless of the presence of inoculum, columns containing nitrate consistently produced more oil than those without nitrate during incubations. Microbial reduction of nitrate to nitrogen with production of carbon dioxide might contribute to the establishment of gas drive. The presence of nitrate could also lead to increased production of biomass and/or biosurfactants, reducing oil-water interfacial tension. Counter-diffusion of nitrogen and carbon dioxide dissolved in column liquids and of helium or argon present in the vial gas phase likely contributed to the establishment of gas drive in all columns. Heptane was present in consistently lower concentrations in columns with nitrate than in those without nitrate at the end of incubations, suggesting that it was an important source of carbon and energy for microbial growth. Azoarcus spp. dominated the consortium when the inoculum was grown with heptane as the sole source of carbon and energy, indicating that this bacterium might contribute to the observed oil production.     

Bacterial adsorption to thermoresponsive polymer surfaces

The ability of microorganisms to `recognise' a change in the hydrophobicity/hydrophilicity balance of the surface was demonstrated using thermoresponsive poly(N-isopropylacrylamide) co-polymers with different Lower Critical Solution Temperatures. The polymers were grafted onto hydrolysed glass under well controlled conditions and the adhesion was followed using ¹³C-labelled Listeria monocytogenes. Attachment of the bacteria was found to be directly affected by the polymer transition from a hydrophilic to a hydrophobic state but by less than one order of magnitude.     

黑曲霉突变株葡萄糖淀粉酶的化学组成及其光谱学性质

黑曲霉突变株葡萄糖淀粉酶中一型GAI舍糖量为17.6%。氨基酸分析表明,天门冬氨酸和谷氨酸(包括酰胺)占20.3%,苏氨酸和丝氨酸占25.1%,三种碱性氨基酸占6%。紫外光谱在278nm和250nm处分别有最大和最小吸收;其荧光光谱的最大激发波长和发射波长分别为284nm与342nm;远紫外CD谱表现为一双负峰;在溶液中的构象是α-螺旋10.6%,β折叠16.3%和无规卷曲73.1%。