Crystal structure of human spermidine/spermine N1-acetyltransferase (hSSAT): the first structure of a new sequence family of transferase homologous superfamily.
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Acetyltransferases↗
Biomedical subjects
Da-Cheng Wang
Publications and source records attributed to Da-Cheng Wang.
Crystallization and preliminary X-ray analysis of the highly thermostable sweet protein Mabinlin II.
Mabinlin II is a thermostable sweet protein isolated from the mature seeds of Capparis masaikai Levl. grown in the subtropical region of the South of China. The Mabinlin II has been crystallized and diffraction data were collected to 1.7 A resolution. The crystal belongs to space group C2 with unit cell parameters a = 80.11 A, b = 51.08 A, c = 47.34 A, beta = 122.77 degrees.
Amino Acid Sequence↗
A periplasmic glutamate/aspartate binding protein from Shigella flexneri: Gene cloning, over-expression, purification and preliminary crystallographic studies of the recombinant protein.
Periplasmic substrate binding proteins (PSBPs) are essential components of the bacterial periplasmic transport system, which transports a wide variety of nutrients from the periplasmic space to the cytoplasm. The glutamate/aspartate binding protein SfGlu/AspBP is a unique PSBP consisting of 279 amino acid residues. The SfGlu/AspBP gene was cloned, over-expressed, and purified by immobilized metal ion affinity chromatography and size-exclusion chromatography. The recombinant protein SfGlu/AspBP has been crystallized by the hanging-drop vapor-diffusion method and its X-ray diffraction data were collected at an atomic resolution of 1.15 A. The crystals belong to the space group P2(1) with unit cell parameters: a = 48.41 A, b = 68.18 A, c = 80.21 A and beta = 98.78 degrees. There are two molecules per asymmetric unit.
Amino Acid Sequence↗
Expression, purification, crystallization and preliminary crystallographic analysis of the human intracellular chloride channel protein CLIC4.
The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method. The crystals diffracted to 2.2 A resolution and were found to belong to space group P121, with unit-cell parameters a = 73.19, b =86.05, c = 73.38 A, beta = 112.99 degrees and three molecule per asymmetric unit.
Chloride Channels↗
Crystal structure of human SH3BGRL protein: the first structure of the human SH3BGR family representing a novel class of thioredoxin fold proteins.
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Amino Acid Sequence↗
Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1.
Scorpion alpha-like toxins are proteins that act on mammalian and insect voltage-gated Na+ channels. Therefore, these toxins constitute an excellent target for examining the foundations that underlie their target specificity. With this motive we dissected the role of six critical amino acids located in the five-residue reverse turn (RT) and C-tail (CT) of the scorpion alpha-like toxin BmK M1. These residues were individually substituted resulting in 11 mutants and were subjected to a bioassay on mice, an electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5 and para), a CD analysis and X-ray crystallography. The results reveal two molecular sites, a couplet of residues (8-9) in the RT and a hydrophobic surface consisting of residues 57 and 59-61 in the CT, where the substitution with specific residues can redirect the alpha-like characteristics of BmK M1 to either total insect or much higher mammal specificity. Crystal structures reveal that the pharmacological ramification of these mutants is accompanied by the reshaping of the 3D structure surrounding position 8. Furthermore, our results also reveal that residues 57 and 59-61, located at the CT, enclose the critical residue 58 in order to form a hydrophobic "gasket". Mutants of BmK M1 that interrupt this hydrophobic surface significantly gain insect selectivity.
Amino Acid Sequence↗
Crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita.
The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.
Agaricales↗
Optimizing protein crystal growth through dynamic seeding.
A dynamic seeding method that is different from the conventional method of seeding drops that have been equilibrated is described. The method basically consists of two steps. Firstly, microseeding was used in association with adjustment of the seeding-drop components, including buffer, additive and concentrations of the precipitants and protein, in order to screen suitable seeding conditions under which microseeds are seeded into a new non-equilibrated drop as the dynamic macroseed drop for the following step. Secondly, after being equilibrated for various times against the reservoir solution, the macroseed drops were used to prepare a dilution series with which the qualified crystals could be harvested using macroseeding. Compared with a conventional seeding technique, this method is distinct with a dynamic situation of macroseed drops before macroseeding and a non-equilibrium serial seeding where all the seeds are seeded into new non-equilibrated drops and the micro/macroseeding are efficiently combined into a whole system. The method simplifies control of the number of microseeds because an excess of microseeds has little effect on the final result. The method also simplifies the manipulation of macroseeds by optimizing the equilibration time and the dilution multiple of the macroseed drops before macroseeding. This dynamic seeding technique has been used in the crystallization of novel protein CutCm, which has a fast crystal-growth rate, and proved that the method is useful for optimizing protein crystallization.
Bacterial Proteins↗
Surface morphology and kinetic properties in rapid growth of EAFP protein crystals investigated by atomic force microscopy.
The whole process of EAFP protein monoclinic crystal growth with an extremely fast rate has been observed by atomic force microscopy. The results showed that the patterns of the growth images in rapidly growing crystals are complicated. The two-dimensional multi-layered stacks of growth steps are characteristic of higher supersaturation and the growth of steps proceeds in a manner of strong anisotropic spiral dislocations dominantly under lower supersaturation conditions. The complex dislocation sources, including multiple dislocation and multi-interacting single dislocation sources, the constant step-split and the propagation of trooped steps were observed on the {100} surfaces of growing EAFP crystals. The step height of each layer generated either by two-dimensional nucleation at higher supersaturation or by screw dislocation at lower supersaturation is about 2-3 nm, which corresponds to the length of the crystallographic unit cell. Although the rate of advancement for each growth step is similar to that of other protein crystal growth, the unique way of the propagation distinct with the trooped steps, by which a bundle of steps are strapped together, would be responsible for the rapid growth of EAFP crystals. All features show a possible mechanism by which the fast growth of EAFP crystals could be attained. The structural basis of the growth mechanism is also discussed.
Eucommiaceae↗
Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution.
The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 A. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.
Cloning, Molecular↗
Crystal structure of the copper homeostasis protein (CutCm) from Shigella flexneri at 1.7 A resolution: the first structure of a new sequence family of TIM barrels.
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Amino Acid Sequence↗
Molecular basis of the mammalian potency of the scorpion alpha-like toxin, BmK M1.
In-depth structure-function studies of voltage-gated Na+ channels and peptide toxins are continuously increasing our understanding of their interaction. In this study, an effective yeast expression system was used to study the role of 14 N- and C-terminal residues from the alpha-like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. With the use of site-directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5, and para), and a circular dichroism analysis. Our results reveal large mutant-dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids, we were able to redirect the alpha-like characteristics of BmK M1 (active on both mammals and insects) to either much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion alpha-like toxin, BmK M1 on voltage-gated Na+ channels.
Animals↗
Structural mechanism governing the quaternary organization of monocot mannose-binding lectin revealed by the novel monomeric structure of an orchid lectin.
Two isoforms of an antifungal protein, gastrodianin, were isolated from two subspecies of the orchid Gastrodia elata, belonging to the protein superfamily of monocot mannose-specific lectins. In the context that all available structures in this superfamily are oligomers so far, the crystal structures of the orchid lectins, both at 2.0 A, revealed a novel monomeric structure. It resulted from the rearrangement of the C-terminal peptide inclusive of the 12th beta-strand, which changes from the "C-terminal exchange" into a "C-terminal self-assembly" mode. Thus, the overall tertiary scaffold is stabilized with an intramolecular beta-sheet instead of the hybrid observed on subunit/subunit interface in all known homologous dimeric or tetrameric lectins. In contrast to the constrained extended conformation with a cis peptide bond between residues 98 and 99 commonly occurring in oligomers, a beta-hairpin forms from position 97 to 101 with a normal trans peptide bond at the corresponding site in gastrodianin, which determines the topology of the C-terminal peptide and thereby its unique fold pattern. Sequence and structure comparison shows that residue replacement and insertion at the position where the beta-hairpin occurs in association with cis-trans inter-conversion of the specific peptide bond (97-98) are possibly responsible for such a radical structure switch between monomers and oligomers. Moreover, this seems to be a common melody controlling the quaternary states among bulb lectins through studies on sequence alignment. The observations revealed a structural mechanism by which the quaternary organization of monocot mannose binding lectins could be governed. The mutation experiment performed on maltose-binding protein-gastrodianin fusion protein followed by a few biochemical detections provides direct evidence to support this conclusion. Potential carbohydrate recognition sites and biological implications of the orchid lectin based on its monomeric state are also discussed in this paper.
Amino Acid Sequence↗
Molecular character of the recombinant antitumor lectin from the edible mushroom Agrocybe aegerita.
The lectin from Agrocybe aegerita (AAL) has been found to possess potent tumor-suppressing function and tumor cell apoptosis-inducing activity. In this paper, we report the full sequence, the active expression of the gene encoding AAL at a high level and bioassay of the binding property with lactose, apoptosis-inducing activity and DNase activity of recombinant AAL (rAAL). The results reveal that AAL is a member of the galectin family and the dimeric form is the active unit for the functional performance. The rAAL showed comparable tumor cell apoptosis-inducing activity with the wild AAL but no DNase activity at all. The molecular characters revealed by this study are significant for the in-depth investigation of the functional mechanism of this interesting protein.
Agaricales↗
Pharmacological comparison of two different insect models using the scorpion alpha-like toxin BmK M1 from Buthus martensii Karsch.
In this study, the effect of the scorpion alpha-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na(+) channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding alpha-like toxins. Moreover, with the aid of the alpha-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.
Animals↗
Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch.
The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.
Animals↗
Crystallization and preliminary crystallographic studies of an antitumour lectin from the edible mushroom Agrocybe aegerita.
An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 A and 3.0 A, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 A, b = 123.98 A, c = 56.86 A, alpha= beta=90 degrees, gamma = 120 degrees and a = 123.69 A, b = 123.69 A, c = 56.64 A, alpha = beta = 90 degrees, gamma = 120 degrees, respectively.
Agaricales↗
Purification and preliminary crystallographic studies of CutC, a novel copper homeostasis protein from Shigella flexneri.
CutC is a novel copper homeostasis protein containing 248 amino acids. Here we report the cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of CutC from Shigella flexneri 2a. Purification of CutC and its selenomethionine (SeMet) derivate were done using immobilized metal ion affinity chromatography, size-exclusion and ion-exchange chromatography. The purified proteins were crystallized using the hanging drop vapor diffusion method. The diffraction data for the native and SeMet CutC, respectively, have been collected with resolution of 1.7 A and 2.1 A. They belong to the space group C2221 and similar cell dimension. The native protein crystals have cell parameters: a=75.3267, b=97.6718, c=132.6910.
Animals↗