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Lirong Chen

Publications and source records attributed to Lirong Chen.

17 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↗
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

Away from the edge II: in-house Se-SAS phasing with chromium radiation.

Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.

Chorismate Mutase↗
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

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↗
PubMed2004

Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease.

Under the guidance of bioassay, the EtOAc extract fraction of the Traditional Chinese Medicine (TCM) Galla Chinese was found to be efficient in inhibiting the NS3 protease of HCV and purified the fraction to get three polyphenol compounds 1,2,6-tri-O-galloyl-beta-D-glucose (1), 1,2,3,6-tetra-O-galloyl-beta-D-glucose (2), and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (3), which were identified as inhibitors of Hepatitis C Virus (HCV) NS3 protease. Compounds 1, 2, and 3 inhibited HCV NS3 protease with IC50 of 1.89, 0.75, and 1.60 microM, respectively.

Antiviral Agents↗
PubMed2004

Separation and screening of compounds of biological origin using molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery in 1972, molecularly imprinted polymers have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. They have been utilized as sensors, catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography, mimics of enzymes, receptors and antibodies. Among which, the application of molecularly imprinted polymers for molecular recognition-based separation and screening of compounds has undergone rapid extension and received much attention in recent years. This article mainly focuses on the separation and screening of certain pharmacophoric compounds of interests from biological origin using molecular imprinting technology. Examples of extraction and recognition of active components as anti-tumors or anti-Hepatitis C virus inhibitors from Chinese traditional herbs using molecularly imprinting technology are particularized in this article. Comparison between the screening effect based on MIPs and that based on antibodies is also represented. Consequently the merits and demerits of these two technologies are highlighted.

Biological Products↗
PubMed2004

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.

Animals↗
PubMed2004

Photodynamic therapy for age-related macular degeneration.

PURPOSE: To evaluate short-term effects of single photodynamic therapy (PDT) for age-related macular degeneration (AMD) accompanied with choroidal neovascularization (CNV). METHODS: We analyzed retrospectively the effects of single PDT for 20 patients (20 eyes) with CNV caused by AMD. Corrected visual acuity, fluorescein angiography (FA) and optic coherence tomography (OCT) were examined before and after PDT. All patients were followed up at least 3 months. RESULTS: At the end of 3-month follow-up, 5 eyes had vision progress, 15 eyes had vision stable and no eye had vision deterioration. Fluorescein angiography one week post PDT showed cessation of fluorescein leakage in 8 eyes with predominant classic CNV, and reduction of fluorescein leakage in 12 eyes with minimal classic CNV or occult CNV without classic component. At the 3-month following PDT fluorescein angiography showed fluorescein leakage reappeared in 4 of 8 eyes with predominant classic CNV. Among 12 eyes with minimal classic CNV or occult CNV without classic component, 9 eyes showed decreased or unchanged fluorescein leakage, 3 eyes had a progression of fluorescein leakage. Optic coherence tomography showed obvious recovery of serous sensory retinal detachment after PDT. CONCLUSION: PDT may occlude or inhibit CNV caused by AMD in short-term. No obvious side effects were noticed.

Aged↗
PubMed2003

Application of a molecularly imprinted polymer for the effective recognition of different anti-epidermal growth factor receptor inhibitors.

A molecularly imprinted polymer (MIP) was prepared using (E)-piceatannol, a natural potential anti-epidermal growth factor receptor (EGFR) inhibitor, as the template and 4-vinylpyridine as the functional monomer. The template was isolated from a Chinese traditional Tibetan medicinal herb, Caragana jubata, by a solid-phase extraction procedure. The crude extract of this herb was loaded on the MIP column for the binding test, and two different compounds besides the template itself were specifically recognized by the polymer, which were identified to be butein and quercetin possessing potent anti-EGFR tyrosine kinase activities with IC(50) values of 10 and 15 microM, respectively. Affinity and selectivity for these inhibitors and another three compounds coexisting with the template in this herb were evaluated in the chromatographic mode. For the first time, the affinity of a molecularly imprinted polymer was investigated to be correlative to the bioactivities of the analytes. The chromatographic behavior of the analytes was consistent with their activity values: the more active inhibitor was retained longer on the MIP. This research work afforded us a new approach for the effective recognition of novel anti-EGFR inhibitors from herbs by using the MIP as the receptor mimic to assay the bioactivities of reserved components, which will be very helpful in the direct separation of lead candidates for anticancer drugs.

Caragana↗
PubMed2003

Frontal affinity chromatography combined on-line with mass spectrometry: a tool for the binding study of different epidermal growth factor receptor inhibitors.

Frontal affinity chromatography (FAC) is a simple but powerful method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of retardation of the elution front. By combination of FAC with a PE-Mariner electrospray ionization mass spectrometry, a very efficient and straightforward procedure was developed herein for analyzing the binding properties of different inhibitors of the epidermal growth factor receptor (EGFR). In this study, a polyclonal antibody prepared with a known anti-EGFR inhibitor coupled with bovine serum albumin was adopted as the stationary phase in the FAC system. Using the antibody to mimic the receptor, other different anti-EGFR inhibitors as well as the small-molecule half-antigen itself were recognized directly from the crude extract of herb, which afforded us a novel promising approach for the efficient screening of lead compounds or drug candidates from natural resources.

Animals↗
PubMed2003

Frontal immunoaffinity chromatography with mass spectrometric detection: a method for finding active compounds from traditional Chinese herbs.

Frontal affinity chromatography (FAC) using immobilized polyclone antibodies of compound A coupled with mass spectrometry was used for the screening of affinity compounds from an extract of Phyllanthus urinaria L. Mass spectrometry was used as an analyzer of FAC. It can analyze the frontal affinity chromatogram of each compound of the extract in one program. The extract was dissolved in 2 mM NH4OAc at a concentration of 10 microg/ mL, then loaded on the immobilized antibody column, and data were collected from mass spectrometry to get a frontal affinity chromatogram. The screening of extract resulted in brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U. Activity analyses give high inhibitory activities to these compounds. This research work afforded us a new approach to find new leading compounds from nature or a man-made combinatorial library that have different structure styles or to find substitutes for the synthetic active compound that has high toxicity.

Animals↗
PubMed2003

Selective extraction of functional components derived from herb in plasma by using a molecularly imprinted polymer based on 2,2-bis(hydroxymethyl)butanol trimethacrylate.

To make molecularly imprinted polymer (MIP) solid-phase extraction (SPE) capable of direct clean-up of aqueous biological samples, an anti-quercetin MIP with evident hydrophobic matrix was synthesized using acrylamide (AA) as the functional monomer and 2,2-bis(hydroxymethyl)butanol trimethacrylate (TRIM) as the crosslinker. The affinity and selectivity were evaluated by liquid chromatography, and the binding sites and the dissolution constants were measured by frontal chromatography. Compared with the AA-co-ethyleneglycol dimethacrylate (EDMA) MIP, the anti-quercetin AA-co-TRIM MIP exhibited stronger binding and possessed improved column efficacy. A linear plot of the peak area versus sample size (in the range of 0.4-2.2 microg) was obtained, which made it promising for the MIP columns to be directly used for analysis. Before MIP-SPE of the sample of plasma, several washing solvents were tested and it was shown that the careful choice of the right washing solvent is the key step to successful sample extraction. The anti-quercetin AA-co-TRIM polymer selectively extracted quercetin, the effective component in the plasma of rats fed the hydrolyzed extract of Gingko biloba L. The recovery (67%) for MIP-SPE was calculated using spiked plasma. The results of the present work showed that the properties of MIP could be improved by modifying the polymerization and that MIP-SPE could be used for direct clean-up of biological samples for the analysis of functional components in vivo originating from an extract of medicinal herbs.

Animals↗
PubMedDate not supplied

Mapping the binding site of a large set of quinazoline type EGF-R inhibitors using molecular field analyses and molecular docking studies.

In the current work, three-dimensional QSAR studies for one large set of quinazoline type epidermal growth factor receptor (EGF-R) inhibitors were conducted using two types of molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). These compounds belonging to six different structural classes were randomly divided into a training set of 122 compounds and a test set of 13 compounds. The statistical results showed that the 3D-QSAR models derived from CoMFA were superior to those generated from CoMSIA. The most optimal CoMFA model after region focusing bears significant cross-validated r(2)(cv) of 0.60 and conventional r(2) of 0.92. The predictive power of the best CoMFA model was further validated by the accurate estimation to these compounds in the external test set, and the mean agreement of experimental and predicted log(IC(50)) values of the inhibitors is 0.6 log unit. Separate CoMFA models were conducted to evaluate the influence of different partial charges (Gasteiger-Marsili, Gasteiger-Hückel, MMFF94, ESP-AM1, and MPA-AM1) on the statistical quality of the models. The resulting CoMFA field map provides information on the geometry of the binding site cavity and the relative weights of various properties in different site pockets for each of the substrates considered. Moreover, in the current work, we applied MD simulations combined with MM/PBSA (Molecular mechanics/Possion-Boltzmann Surface Area) to determine the correct binding mode of the best inhibitor for which no ligand-protein crystal structure was present. To proceed, we define the following procedure: three hundred picosecond molecular dynamics simulations were first performed for the four binding modes suggested by DOCK 4.0 and manual docking, and then MM/PBSA was carried out for the collected snapshots. The most favorable binding mode identified by MM/PBSA has a binding free energy about 10 kcal/mol more favorable than the second best one. The most favorable binding mode identified by MM/PBSA can give satisfactory explanation of the SAR data of the studied molecules and is in good agreement with the contour maps of CoMFA. The most favorable binding mode suggests that with the quinazoline-based inhibitor, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 766, not Thr 830 as proposed by Wissner et al. (J. Med. Chem. 2000, 43, 3244). The predicted complex structure of quinazoline type inhibitor with EGF-R as well as the pharmacophore mapping from CoMFA can interpret the structure activities of the inhibitors well and afford us important information for structure-based drug design.

Binding Sites↗