Biomedical subjects

Yuhe Liang

Publications and source records attributed to Yuhe Liang.

6 recordsLinked to original sources

PubMed2006

A large-scale, high-efficiency and low-cost platform for structural genomics studies.

A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US 10,000 dollars.

Bacillus subtilis↗

PubMed2005

Alternative IMP binding in feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Crystal structures of Thermoanaerobacter tengcongensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) apoenzyme and the enzyme-inosine monophosphate (IMP) complex have been determined to 2.5A and 2.2A resolution, respectively. The active form of the enzyme was identified as a tetramer in solution and the K(i) value of IMP was measured to be 45 microM for alpha-D-phosphoribosyl-1-pyrophosphate (PRPP). Conformation of the flexible loop in T.tengcongensis HGPRT, which is involved in substrate PRPP binding, is different from that observed in phosphoribosyltransferases (PRTs). It contains a 3-10 helix, and a unique double serine repeat. This loop is ordered even in the apoenzyme and assumes a half-closed conformation. The primary magnesium ion is directly coordinated by side-chains of Glu101 and Asp102, and water molecules in the apoenzyme, suggesting a possible prerequisite role for substrate PRPP binding. Most interestingly, an alternative IMP binding mode is found in the structure of T.tengcongensis HGPRT-IMP complex. The 5'-phosphate of IMP occupies the PPi position usually seen in PRT-PRPP complexes. This new observation is consistent with the lower K(i) value of IMP and may suggest a mechanism involving multiple modes of interactions between IMP and T.tengcongensis HGPRT in product release and feedback inhibition. The structure of T.tengcongensis HGPRT is compared with those of mesophilic HPRTs, and several possible features contributing to its thermostability are elucidated. Overall, T.tengcongensis HGPRT appears to be more diverged from other PRTs.

Amino Acid Sequence↗

PubMed2005

The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus.

Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-A resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.

Active Transport, Cell Nucleus↗

PubMed2004

Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.

The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

Base Sequence↗

PubMed2004

Protein preparation, crystallization and preliminary X-ray analysis of human adrenal gland protein AD-004.

The adrenal gland protein AD-004 was identified in the human adrenal gland. Full-length AD-004 contains 172 amino acids, with a predicted molecular weight of about 20 kDa. In attempts to crystallize human AD-004, the gene was subcloned into a modified pET vector, pET21-DEST, with an N-terminal His(5) tag using the Gateway cloning system, followed by protein expression in Escherichia coli strain BL21(DE3). The protein was purified in two steps to near-homogeneity and was crystallized. The crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 99.56, c = 57.19 A. A complete 2.0 A data set has been collected at a rotating-anode X-ray source and structure determination is under way.

Adrenal Glands↗

PubMed2003

Protein preparation, crystallization and preliminary X-ray crystallographic studies of a thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Native and His-tagged mutant (L160I) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned, expressed in Escherichia coli and purified. Both proteins were crystallized with polyethylene glycol as the main precipitant at 293 K using the hanging-drop vapour-diffusion method. The crystal of native HGPRT belongs to space group C222(1), with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 A, and diffracted to 2.2 A resolution on an in-house X-ray generator. The crystal of the His-tagged mutant (L160I) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 A, and diffracted to 1.7 A resolution in-house.

Bacillaceae↗