Houming Wu

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimers disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein‐3 (MT‐3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the α‐domain of human MT‐3 (residues 32–68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT‐3 shows two metal–thiolate clusters, one in the N‐terminus (β‐domain) and one in the C‐terminus (α‐domain). The overall fold of the α‐domain is similar to that of mouse MT‐3. However, human MT‐3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT‐3. Surprisingly, the backbone dynamics of the protein revealed that the β‐domain exhibits similar internal motion to the α‐domain, although the N‐terminal residues are more flexible. Our results may provide useful information for understanding the structure–function relationship of human MT‐3.

Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.

Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

Solution structure of all parallel G-quadruplex formed by the oncogene RET promoter sequence

RET protein functions as a receptor-type tyrosine kinase and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the promoter plays an important role in the transcriptional regulation of RET. Here, we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. The overall G-quadruplex is composed of three stacked G-tetrad and four syn guanines, which shows distinct features for all parallel-stranded folding topology. The core structure contains one G-tetrad with all syn guanines and two other with all anti-guanines. There are three double-chain reversal loops: the first and the third loops are made of 3 nt G-C-G segments, while the second one contains only 1 nt C10. These loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure and their conformations are in accord with the experimental mutations. The distinct RET promoter G-quadruplex structure suggests that it can be specifically involved in gene regulation and can be an attractive target for pathway-specific drug design.

Spectroscopic Properties of QSOs Selected from Ultraluminous Infrared Galaxy Samples

We performed spectroscopic observations for a large infrared quasi-stellar object (QSO) sample with a total of 25 objects. The sample was compiled from the QDOT redshift survey, the 1 Jy ultraluminous IRAS galaxy survey, and a sample obtained by a cross-correlation study of the IRAS Point-Source Catalogue with the ROSAT All-Sky Survey Catalogue. Statistical analyses of the optical spectra show that the vast majority of infrared QSOs have narrow permitted emission lines (with FWHM of Hβ less than 4000 km s-1) and more than 60% of them are luminous narrow-line Seyfert 1 galaxies. Two of the infrared QSOs are also classified as low-ionization broad absorption line (lo-BAL) QSOs. More than 70% of infrared QSOs are moderately or extremely strong Fe II emitters. This is the highest percentage of strong Fe II emitters in all subclasses of QSO Seyfert 1 samples. We found that the Fe II to Hβ line ratio is significantly correlated with the [O III] λ5007 peak and Hβ blueshift. Soft X-ray–weak infrared QSOs tend to have large blueshifts in permitted emission lines and significant Fe II 48, 49 (5100–5400 A) residuals relative to the Boroson & Green Fe II template. If the blueshifts in permitted lines are caused by outflows, then they appear to be common in infrared QSOs. As the infrared-selected QSO sample includes both luminous narrow-line Seyfert 1 galaxies and lo-BAL QSOs, it could be a useful laboratory to investigate the evolutionary connection among these objects.

Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

Converting Cytochrome b5 into Cytochrome c‐Like Protein

Cytochrome b5 (cyt b5) is a well-studied b-type cytochrome with heme bound noncovalently. Its heme-binding ability depends mostly on the strong axial ligation provided by residues His63 and His39 (Figure 1). Mutation studies on the axial ligands, aimed at creating new proteins with novel catalytic reactivity, have been limited by the fact that such alteration would lead to a substantial decrease in the heme-binding stability. Therefore, it would be of great interest to construct covalent linkages between heme and polypeptides so as to stabilize the heme prosthetic group, as found in c-type cytochromes, in which heme is covalently attached to a polypeptide chain via two thioether bonds between the heme vinyl groups and the cysteine residues of a classic Cys-Xaa-Xaa-CysHis (CXXCH) heme-binding peptide motif in the protein. By converting cyt b5 into a cyt c-like protein, it will be much easier to fulfill the functional conversion of cyt b5 mentioned above. Based on the wealth of molecular information obtained from X-ray crystallography studies of cyt b5 (PDB entry 1CYO), [5] we found out that residues Asn57 and Ser71 are close to the

A new triterpenoid from Entodon okamurae Broth.

One new triterpenoid, entokamurol (1), was isolated from Entodon okamurae Broth, together with other nine compounds, namely dryocrassol (2), chrysophamol (3), physcion (4), 10-nonacosamnol (5), n-hexadecanol (6), phthalic acid isodibutyl ester (7), curcumol (8), β-sitosterol (9) and daucosterol (10). Their structures were elucidated on the basis of extensive NMR (DEPT, DQF-COSY, HMQC, HMBC and NOESY), IR and MS studies. All the compounds were isolated and identified from the genus of Entodon for the first time, and it is also the first report of a guaiane-type sesquiterpenoid and compounds with anthraquinone skeleton in mosses.

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily α‐KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole‐cell patch‐clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two‐dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common α/β‐motif, but showed distinct local conformation in the loop between α‐helix and β‐sheet in comparison with typical short‐chain scorpion toxins (e.g., CTX and NTX). Also, the α helix is shorter and the β‐sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the α‐helix and β‐sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a α‐mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter‐residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter‐residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor. Proteins 2004.

Purification, characterization and sequence determination of BmKK4, a novel potassium channel blocker from Chinese scorpion Buthus martensi Karsch.

The scorpion neurotoxin BmKK4 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange and reversed phase chromatography. The primary sequence of BmKK4 was determined using the tandem MS/MS technique and the cDNA database searching as followings: ZTQCQ SVRDC QQYCL TPDRC SYGTC YCKTT (NH(2)). BmKK4 is the first isolated member of a new subfamily alpha-KTx17 of scorpion K(+) toxins.