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Bi-Cheng Wang

Publications and source records attributed to Bi-Cheng Wang.

At least 19 recordsLinked to original sources
PubMed2007

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica.

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.

Amino Acid Sequence↗
PubMed2006

Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus.

The gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2(1), with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 A, beta = 104.9 degrees; the crystal diffracted to a resolution of 1.9 A. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 A, and diffracted to 1.75 A resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2(1) and R32, respectively.

Coenzymes↗
PubMed2006

Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state.

The crystal structure at 1.93-A resolution is determined for the Ca2+-discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound Ca2+ and/or coelenteramide. Among these structures, global conformational changes are small, typical of the class of "calcium signal modulators" within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.

Amides↗
PubMed2005

Crystal structures of tyrosyl-tRNA synthetases from Archaea.

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.

Adenosine Monophosphate↗
PubMed2005

Reconstruction of ancient genome and gene order from complete microbial genome sequences.

Microbial genome sequences provide us with the fossil records for inferring their origination and evolution. Assuming that current microbial genomes are the evolutionary results of ancient genomes or fragments and the neighboring genes in ancient genomes are more likely neighbors in current genomes, in this paper we proposed a paleontological algorithm and assembled the orthologous gene groups from 66 complete and current microbial genome sequences into a pseudo-ancient genome, which consists of continuous fragments of various sizes. We performed bootstrap resampling and correlation analyses and the results showed that the assembled ancient genome and fragments are statistically significant and the genes of the same fragment are inherently related and likely derived from common ancestors. This method provides a new computational tool for studying microbial genome structure and evolution.

Algorithms↗
PubMed2005

The high-throughput protein-to-structure pipeline at SECSG.

Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.

Crystallization↗
PubMed2005

Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoacetica.

A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.

Acetyl Coenzyme A↗
PubMed2005

Parameter-space screening: a powerful tool for high-throughput crystal structure determination.

The determination of protein structures on a genomic scale requires both computing capacity and efficiency increases at many stages along the complex process. By combining bioinformatics workflow-management techniques, cluster-based computing and popular crystallographic structure-determination software packages, an efficient and powerful new tool for structural biology/genomics has been developed. Using the workflow manager and a simple web interface, the researcher can, in a few easy steps, set up hundreds of structure-determination jobs, each using a slightly different set of program input parameters, thus efficiently screening parameter space for the optimal input-parameter combination, i.e. a set of parameters that leads to a successful structure determination. Upon completion, results from the programs are harvested, analyzed, sorted based on success and presented to the user via the web interface. This approach has been applied with success in more than 30 cases. Examples of successful structure determinations based on single-wavelength scattering (SAS) are described and include cases where the 'rational' crystallographer-based selection of input parameters values had failed.

Computational Biology↗
PubMed2005

Structure of the conserved transcriptional repressor enhancer of rudimentary homolog.

erh (enhancer of rudimentary homolog) is a ubiquitously expressed transcriptional coregulator that is highly conserved among eukaryotes, from humans to plants to protozoa. Functions attributed to erh include enhancement of pyrimidine biosynthesis, a role in cell cycle regulation, and repression of the tissue-specific transcription factor HNF-1 (hepatocyte nuclear factor-1) through binding the coactivator DCoH (dimerization cofactor of HNF1). No homologous sequences, other than erh orthologs, have been identified, and little is known about the interactions of erh. To further elucidate its function, we determined the crystal structure of erh to 2.0 A resolution. The erh structure is a novel alpha + beta fold consisting of a four-stranded antiparallel beta sheet with three amphipathic alpha helices situated on one face of the beta sheet. Structure-based searches of the Protein Data Bank, like sequence-based searches, failed to identify paralogs. We present structural and biochemical evidence that erh functions as a dimer. The dimer interface consists of a beta sandwich composed of the beta sheet from each monomer. Many of the surface residues of erh are conserved, including patches of hydrophobic and charged residues, suggesting protein-protein interaction interfaces. Two putative CKII phosphorylation sites are highly ordered in the structure and are predicted to disrupt dimerization and protein-protein interactions.

Amino Acid Sequence↗
PubMed2005

Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.

Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel (3)S(1) sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the (1)C(4) conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the -1 subsite. A novel (3)H(4) conformation is proposed as the exploded transition state.

Amino Acid Substitution↗
PubMed2005

All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin.

The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hyroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with product, coleneteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.

Aequorin↗
PubMed2005

Protein production and crystallization at SECSG -- an overview.

Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins, which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.

Animals↗
PubMed2005

A test of enhancing model accuracy in high-throughput crystallography.

The high throughput of structure determination pipelines relies on increased automation and, consequently, a reduction of time spent on interactive quality control. In order to meet and exceed current standards in model accuracy, new approaches are needed for the facile identification and correction of model errors during refinement. One such approach is provided by the validation and structure-improvement tools of the MOL: PROBITY: web service. To test their effectiveness in high-throughput mode, a large subset of the crystal structures from the SouthEast Collaboratory for Structural Genomics (SECSG) has used protocols based on the MOL: PROBITY: tools. Comparison of 29 working-set and 19 control-set SECSG structures shows that working-set outlier scores for updated Ramachandran-plot, sidechain rotamer, and all-atom steric criteria have been improved by factors of 5- to 10-fold (relative to the control set or to a Protein Data Bank sample), while quality of covalent geometry, R(work), R(free), electron density and difference density are maintained or improved. Some parts of this correction process are already fully automated; other parts involve manual rebuilding of conformations flagged by the tests as trapped in the wrong local minimum, often altering features of functional significance. The ease and effectiveness of this technique shows that macromolecular crystal structures from either traditional or high-throughput determinations can feasibly reach a new level of excellence in conformational accuracy and reliability.

Crystallography, X-Ray↗
PubMed2005

Salvaging Pyrococcus furiosus protein targets at SECSG.

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.

Archaeal Proteins↗
PubMed2005

Life in the fast lane for protein crystallization and X-ray crystallography.

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Crystallization↗
PubMed2004

The hyperthermophile protein Sso10a is a dimer of winged helix DNA-binding domains linked by an antiparallel coiled coil rod.

Sso10a is a member of a group of DNA-binding proteins thought to be important in chromatin structure and regulation in the hyperthermophilic archaeon Sulfolobus solfataricus. We have determined the structure of Sso10a to 1.47A resolution directly with unlabelled native crystals by a novel approach using sulfur single-wavelength anomalous scattering (SAS) from a chromium X-ray source. The 95 amino acid residue protein contains a winged helix DNA-binding domain with an extended C-terminal alpha-helix that leads to dimerization by forming a two-stranded, antiparallel coiled-coil rod. The winged helix domains are at opposite ends of the extended coiled coil with two putative DNA-recognition helices separated by 55A and rotated by 83 degrees. Formation of stable dimers in solution is demonstrated by both analytical ultracentrifugation and differential scanning calorimetry. With a T0 of 109 degrees C, Sso10a is one of the most stable two-stranded coiled coils known. The coiled coil contains a rare aspartate residue (D69) in the normally hydrophobic d position of the heptad repeat, with two aspartate-lysine (d-g') interhelical ion pairs in the symmetrical dimer. Mutation of D69 to alanine resulted in an increase in thermal stability, indicating that destabilization resulting from the partially buried aspartate residue cannot be offset by ion pair formation. Possible DNA-binding interactions are discussed on the basis of comparisons to other winged helix proteins. The structure of Sso10a provides insight into the structures of the conserved domain represented by COG3432, a group of more than 20 hypothetical transcriptional regulators coded in the genomic sequences of both crenarchaeota and euryarchaeota.

Amino Acid Sequence↗