重金属结合肽（蛋白）的结构分析

金属离子与金属结合肽（蛋白）的相互作用与应用研究,一直是生物无机化学的重点和热点,也是分子间相互作用研究领域的难点。本研究利用ClustalX、BLAST等生物信息技术与方法对大量已知的重金属结合肽进行分析与数据挖掘。确定筛选获得的重金属结合肽常富含His,无Cys,无金属结合肽模式序列,进化不保守;部分氨基酸序列结构（如六肽）可在蛋白数据库中找到相似序列。序列特征主要为Zn^2＋相关的转录因子。本研究为重金属结合蛋白-重金属离子的相互作用分析简化为重金属结合肽-重金属离子的结构模拟与分析提供了重要的理论基础和研究手段。     

Interaction of the two soluble metal-binding domains of yeast Ccc2 with copper(I)-Atx1

Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second. Similar results are found in ATPases from other organisms and may represent a general feature, whose biochemical implications are not yet fully appreciated.     

Structural variety and stereochemical features of bis(diphenylphosphinyl)[3]ferrocenophane gold(I) complexes

The P,N-[3]ferrocenophane ligand 3 forms a (κP-ligand)AuCl complex (5) upon treatment with (Me₂S)AuCl. The corresponding P,P-[3]ferrocenophane system 4 yields a binuclear (κP,κP-chelate ligand)(AuCl)₂ complex (6) when reacted with 2 equivalents of the (Me₂S)AuCl reagent. Complex 6 features an intramolecular aurophilic Au?Au interaction. Treatment of the P,P-[3]ferrocenophane 4 with 1.0 equiv. of (PPh₃)AuCl gives the tetra-coordinated mono-gold(I) complex (P,P-ligand)(PPh₃)AuCl (7), whereas the cationic [(P,P-ligand)₂Au]⁺[Cl⁻] system is obtained from 4 and 0.5 equivalents of (Me₂S)AuCl. The [(P,P-ligand)₂Au]⁺ system is obtained in different diastereoisomeric forms (8 and 9) depending on the stereochemistry of the pair of P,P-[3]ferrocenophane chelate ligand used. Examples of the complexes 5, 6, 7 and 8 were characterized by X-ray diffraction.     

Chelation of hypocrellin B with zinc ions with electron paramagnetic resonance (EPR) evidence of the photodynamic activity of the resulting chelate

Hypocrellin B (HB), a perylenequinone derivative, is an efficient phototherapeutic agent. The chelation of HB with Zinc ions (Zn²⁺) results in a metal chelate (Zn-HB) which exhibits considerable absorption (λ_max = 612nm) in the phototherapeutic window. The structure of this chelate has been characterized by UV-Vis, IR and mass spectra. The redox potentials of the Zn-HB chelate were E_ox = +1.1V (vs. SCE) and E_re = -0.7V (vs. SCE) as measured using the circle volt curve. The quantum yield of singlet oxygen generated by the Zn-HB chelate was 0.86, which both the electron spin trap (EPR) method and the chemical trap method show to be about 0.1 higher than that of its parent compound HB. In irradiated oxygen-saturated solutions of Zn-HB chelate, superoxide radical anions and hydroxyl radicals were detected by EPR spectroscopy using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agent.     

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.     

Structural studies in solution and in the solid state on the zinc chelate of 2-hydroxy-(4-methylthio)butanoic acid, an effective mineral supplement in animal feeding

The bis-chelate complex of Zn²⁺ with 2-hydroxy-(4-methylthio)butanoate (MHA_-H the anion derived from the so-called methionine hydroxy-analogue, MHA) is an effective, bioavailable mineral supplement for animal feeding. It can be obtained in two solid forms: the anhydrous [Zn(OC(O)CH₂CH(OH)CH₂CH₂SCH₃)₂] and the corresponding dihydrate species, both well distinguishable by IR spectroscopy and powder X-ray diffraction. The crystal and molecular structure of the dihydrate form has been solved by single-crystal X-ray diffraction. It consists of dinuclear bis-chelate species with a bridging carboxylate group, both zinc atoms displaying hexacoordination involving all the hydroxyl and carboxyl groups from the four MHA_-H anions and three oxygens from different water molecules. The fourth water molecule does not participate in coordination. Therefore, the dihydrate complex must be formulated as [Zn₂(OC(O)CH₂CH(OH)CH₂CH₂SCH₃)₄(H₂O)₃] · H₂O (1). A molecular computational analysis has been carried out by density functional theory (DFT) on three possible MHA_-H zinc chelates, i.e. the dinuclear bis-chelate observed in the solid state, the mononuclear bis-chelate diaquo-complex, and the monochelate tetraaquo-complex. Calculations have suggested that between the dinuclear and mononuclear bis-chelates, the preferred form in aqueous solution may be the second one. Moreover, both ¹H (chemical shifts and relaxation rates) and ¹³C NMR data provide further evidence for the formation of Zn/MHA_-H chelates in solution.     

Adjustments of the oxygen diffusing capacity to energetic demands during the development of the quail (Coturnix coturnix japonica)

One of the hypotheses that attempt to explain physiological limitations of energy budgets is the symmorphosis hypothesis, which proposes that if matching structures to functional needs were combined with the strict economy of energy and materials, the result would be an optimal organ design for the specific function it serves. Evidence in favor of symmorphosis in adults is as abundant as evidence against it, but the plasticity of some morphological traits may be dependent on the ontogenetic stage at which acclimation acts. Thus, here we studied the adjustment of structure and function in lungs at different stages of development in the quail Coturnix coturnix japonica under two thermal regimes. Our main results show that i) resting metabolic rate, maximum thermogenic oxygen consumption and oxygen diffusion capacity did not exhibit developmental plasticity for two thermal environments; and ii) oxygen diffusion capacity fully adjusted to resting metabolic rate and maximum oxygen consumption during development. C. coturnix has a low safety factor close to 1 which is consistent with the symmorphosis hypothesis.     

Structural and mechanistic studies on carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily catalyzing the first step in carbapenem biosynthesis

The first step in the biosynthesis of the medicinally important carbapenem family of beta-lactam antibiotics is catalyzed by carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily. CarB catalyzes formation of (2S,5S)-carboxymethylproline [(2S,5S)-t-CMP] from malonyl-CoA and l-glutamate semialdehyde. In addition to using a cosubstrate, CarB catalyzes C-C and C-N bond formation processes as well as an acyl-coenzyme A hydrolysis reaction. We describe the crystal structure of CarB in the presence and absence of acetyl-CoA at 2.24 A and 3.15 A resolution, respectively. The structures reveal that CarB contains a conserved oxy-anion hole probably required for decarboxylation of malonyl-CoA and stabilization of the resultant enolate. Comparison of the structures reveals that conformational changes (involving His(229)) in the cavity predicted to bind l-glutamate semialdehyde occur on (co)substrate binding. Mechanisms for the formation of the carboxymethylproline ring are discussed in the light of the structures and the accompanying studies using isotopically labeled substrates; cyclization via 1,4-addition is consistent with the observed labeling results (providing that hydrogen exchange at the C-6 position of carboxymethylproline does not occur). The side chain of Glu(131) appears to be positioned to be involved in hydrolysis of the carboxymethylproline-CoA ester intermediate. Labeling experiments ruled out the possibility that hydrolysis proceeds via an anhydride in which water attacks a carbonyl derived from Glu(131), as proposed for 3-hydroxyisobutyryl-CoA hydrolase. The structural work will aid in mutagenesis studies directed at altering the selectivity of CarB to provide intermediates for the production of clinically useful carbapenems.     

Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-d-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 Å resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded β-sheet flanked by two α-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons α to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.     

Direct estimation of the oxygen requirements of Achromobacter xylosoxidans for aerobic degradation of monoaromatic hydrocarbons (BTEX) in a bioscrubber

The O₂ requirements for biomass production and supplying maintenance energy demands during the degradation of both benzene and ethylbenzene by Achromobacter xylosoxidans Y234 were measured using a newly proposed technique involving a bioscrubber. Using this approach, relevant microbial parameter estimates were directly and simultaneously obtained via linear regression of pseudo steady-state data. For benzene and ethylbenzene, the biomass yield on O₂, , was estimated on a cell dry weight (CDW) basis as 1.96 ±.25 mg CDW mgO₂ and 0.98 ±.17 mg CDW mgO₂, while the specific rate of O₂ consumption for maintenance, , was estimated as 0.041 ±.008 mgO₂ mg CDW^h and 0.053 ±.022 mgO₂ mg CDW^h, respectively.     

Seasonal changes in the oxygen storage capacity and aerobic dive limits of the muskrat (Ondatra zibethicus)

Summary The oxygen storage capacity and partitioning of body oxygen reserves were compared in summer-and winter-acclimatized muskrats (Ondatra zibethicus). Blood volume, blood oxygen capacity, and skeletal muscle myoglobin content were higher in December than in July (P<0.02). Total lung capacity increased only slightly in winter (P>0.05). The oxygen storage capacity of a diving muskrat was calculated at 25.2 ml O₂ STPD · kg^-1 in July, compared to 35.7 ml O₂ STPD · kg^-1 in December. Blood comprised the major storage compartment in both seasons, accounting for 57% and 65% of the total oxygen stores in summer and winter, respectively. Based on available oxygen stores and previous estimates of the cost of diving, the aerobic dive limit (ADL) increased from 40.9 s in July to 57.9 s in December. Concurrent behavioral studies suggested that most voluntary diving by muskrats is aerobic. However, the proportion of dives exceeding the calculated ADL of these animals was shown to vary with the context of the dive. Only 3.5% of all dives initiated by muskrats floating in the water exceeded their estimated ADL. Provision of a dry resting site and access to a submerged food source increased this proportion to 18–61%, depending on the underwater distance that foraging muskrats were required to swim. Serial dives exceeding the estimated ADL were not accompanied by extended postdive recovery periods.Abbreviations ADL acrobic dive limit - Hb hemoglobin - Hct hematocrit - Mb myoglobin - PaO₂ arterial O₂ tension - STPD standard temperature and pressure, dry     

The effect of adrenaline and high Ca2+ on the mechanical performance and oxygen consumption of the isolated perfused trout (Oncorhynchus mykiss) heart

In heart muscle from mammals, catecholamines frequently evoke an oxygen waste and reduce efficiency. It was examined if this also applies to fish in which heart muscle activity is often restricted by oxygen availability. In the isolated perfused heart from rainbow trout, adrenaline (0.5 microM) increased power output by approximately 97%, when afterload was adjusted to maximum both before and after adrenaline addition, and by approximately 68%, when afterload remained at the maximum obtained before adrenaline addition. Oxygen consumption was enhanced by a similar amount (approximately 70%) in both situations. Hence, efficiency, i.e. power output/oxygen consumption, increased significantly from 25 to 30% for the heart always exposed to maximal afterload, whereas it stayed unchanged at 24% for the heart exposed to control afterload only. Adrenaline increases the Ca2+ activity participating in activation, but doing this by elevating perfusate Ca2+ from 1.5 to 5 mM instead of applying adrenaline did not change the mechanical efficiency, although afterload was maximized. The results suggest that catecholamines enhance heart pump activity in the living trout without any oxygen waste. On the contrary, the oxygen budget may even improve when the peripheral resistance is increased by catecholamines.     

Synthesis and characterization of thiocyanato and chlorodioxo tungsten(VI) compounds: Comparative oxygen atom transfer capability with molybdenum analogs

Four new tungsten-oxo(VI) complexes have been synthesized, characterized spectroscopically and their molecular structure established by X-ray diffraction analysis. These bear identical environment as previously reported molybdenum-oxo(VI) complexes, which allowed direct comparison of their spectroscopic properties. Their capability as oxygen atom transfer agents was found to be significantly lower than their molybdenum analogs.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.     

Electrocatalytic reduction of protons and oxygen at glassy carbon electrodes coated with a cobalt(II)/platinum(II) porphyrin

The electrocatalytic reduction of protons in 1.0 M perchloric acid at glassy carbon electrodes anodically modified with a Co(II)/Pt(II) porphyrin show shifts of 400 mV versus Ag/AgCl when compared to the same electrodes which have not been anodically modified. Anodic cycling of glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin in this study form stable electroactive films capable of improving both electroreduction of protons to hydrogen and oxygen to both peroxide and water. Electrooxidation of glassy carbon electrodes coated with the free base porphyrin show no improvement in catalytic ability for the reduction of protons in acidic solution or the reduction of molecular oxygen in basic solution. Glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin indicate, by rotating disk electrochemistry, that the electrocatalysis of oxygen is a two electron process leading to the formation of hydrogen peroxide. Koutecky-Levich plots of the data obtained from the reduction of oxygen at electrode surfaces coated with the Co(II)/Pt(II) porphyrin after oxidation of the surface indicate that 25% of the oxygen is reduced by four electrons directly to water while 75% of the oxygen is reduced by two electrons to hydrogen peroxide.