Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH

We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the N-15 and H-1 chemical shift of a residue ('i') with those of its immediate N-terminal (i - 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on C-13(beta) chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal H-1 relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.

Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface alpha 5 beta 1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 x 10(8)) and Tomlinson J (Library size 1.37 x 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

Photo-induced double-strand DNA and site-specific protein cleavage activity of L-histidine (mu-oxo)diiron(III) complexes of heterocyclic bases

Three oxo-bridged diiron(III) complexes of L-histidine and heterocyclic bases [Fe-2(mu-O)(L-his)(2)(B)(2)](ClO4)(2) (1-3), where B is 2,2'-bipyridine (bpy),1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized. The bpy complex 1 was structurally characterized by X-ray crystallography. The molecular structure showed a {Fe-2(mu-O)} core in which iron(III) in a FeN4O2 coordination is bound to tridentate monoanionic L-histidine and bidentate bpy ligands. The Fe center dot center dot center dot Fe distance is similar to 3.5 angstrom. The Fe-O-Fe unit is essentially linear, giving a bond angle of similar to 172 degrees. The complexes showed irreversible cyclic voltammetric cathodic response near -0.1 V vs. SCE in H2O-0.1 M KCl. The binuclear units displayed antiferromagnetic interaction between two high-spin (S = 5/2) iron(III) centers giving a -J value of -110 cm(-1). The complexes showed good DNA binding propensity giving a binding constant value of similar to 10(5) M-1. Isothermal titration calorimetric data indicated single binding mode to the DNA. The binding was found to be driven by negative free energy change and enthalpy. The dpq complex 3 showed oxidative double-strand DNA cleavage on exposure to UV-A and visible light. The phen complex 2 displayed single-strand photocleavage of DNA. The DNA double-strand breaks were rationalized from theoretical molecular docking calculations. Mechanistic investigations showed formation of hydroxyl radicals as the reactive species through photodecarboxylation of the L-histidine ligand. The complexes exhibited good binding propensity to bovine serum albumin (BSA) protein in Tris-HCl/NaCl buffer medium. The dpq complex 3 showed UV-A light-induced site-specific oxidative BSA cleavage forming fragments of similar to 45 kDa and similar to 20 kDa molecular weights via SOH pathway.

Modular design of synthetic protein mimics. Crystal structures, assembly, and hydration of two 15- and 16-residue apolar, leucyl-rich helical peptides

Two IS- and 16-residue peptides containing a-aminoisobutyric acid (Aib) have been synthesized, as part of a strategy to construct stereochemically rigid peptide helices, in a modular approach to design of protein mimics. The peptides Boc-(Val-Ala-Leu-Aib),-OMe ( I ) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-(Val-Ala-Leu-Aib()11z)- OhaMvee been crystallized.Both crystals are stable only in the presence of mother liquor or water. The crystal data are as follows. I: C78H140N16019~2H20,P2,, a = 16.391 (3) A, b = 16.860 (3) A, c = 18.428 (3) A, p = 103.02 (I)O, Z = 2, R = 9.6% for 3445 data with lFol >30(F), resolution 0.93 A. 11: C7,Hl,,N,S018.7.5H,0, C2221, a = 18.348 ( 5 ) A, b = 47.382 (1 1) A, c = 24.157 ( 5 ) A, Z =8, R = l0,6%, for 3147 data with lFol > 3a(F), resolution 1.00 A. The 15-residue peptide (11) is entirely a helical, while the 16-residue peptide ( I ) has a short segment of 310 helix at the N terminus. The packing of the helices in the crystals is rather incfficicnt with no particular attractions between Leu-Leu side chains, or any other pair. Both crystals have fairly large voids, which are filled with water molecules in a disordered fashion. Water molecule sites near the polar head-to-tail regions are well detcrmined, those closer to the hydrophobic side chains less so and a number of possible water sites in the remaining "empty" space are not determined. No interdigitation of Leu side chains is observed in the crystal as is hypothesized in the "leucine zipper" class of DNA binding proteins.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of δ-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the ‘derepression’ of δ-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs

An analysis of the recently reported cDNA derived amino acid sequences of mouse (Kleene and Flynn, J. Biol. Chem. , 17272–17277, 1987) and rat (Luersson Image ,Nucl. Acids Res. Image , 3585, 1989). TP2 has revealed the presence of two potential zinc finger motifs involving cysteine and histidine residues. TP2, as purified from rat elongating spermatids, is shown here to contain 0.2 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. On incubation with 10 μM ZnCl2, Image , and subsequent exhaustive dialysis, TP2 had 2 atoms of zinc bound per molecule. The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, Image , with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA. The zinc finger domains of TP2 may play an important role in initiation of chromatin condensation and /or cessation of transcriptional activity during mammalian spermiogenesis. DTT, Dithiothreitol; IAF, Iodoacetamido-fluorescein; SDS, Sodium dodecyl sulfate; PAGE, Polyacrylamide gel electrophoresis; PMSF, Phynyl methyl sulfonyl fluoride

DNA-induced conformational changes in RecA protein. Evidence for structural heterogeneity among nucleoprotein filaments and implications for homologous pairing

We have used circular dichroism as a probe to characterize the solution conformational changes in RecA protein upon binding to DNA. This approach revealed that RecA protein acquires significant amounts of alpha-helix upon interaction with DNA. These observations, consistent with the data from crystal structure (Story, R. M., Weber, I., and Steitz, T. (1992) Nature 355, 318-325), support the notion that some basic domains including the DNA binding motifs of RecA protein are unstructured and might contribute to the formation of alpha-helix. A comparison of nucleoprotein filaments comprised of RecA protein and a variety of DNA substrates revealed important structural heterogeneity. The most significant difference was observed with poly(dG). poly(dC) and related polymers, rich in GC sequences, which induced minimal amounts of alpha-helix in RecA protein. The magnitude of induction of alpha-helix in RecA protein, which occurred concomitant with the production of ternary complexes, was 2-fold higher with homologous than heterologous duplex DNA. Most importantly, the stimulation of ATP hydrolysis by high salt coincided with that of the induction of alpha-helix in RecA protein. These conformational differences provide a basis for thinking about the biochemical and structural transitions that RecA protein experiences during the formal steps of presynapsis, recognition, and alignment of homologous sequences.

A network representation of protein structures: Implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of similar to 3.5 angstrom in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

The protein components of elastoidin

Not all elastoidin samples from different species of sharks show a high content of tryptophan in contrast to one specimen from an unidentified species examined in our earlier study. There may be considerable species-dependent variation in tryptophan content and analytical artefacts may have occurred. Available analyses for tyrosine in different specimens of fibres also suggest the possibility that the variation in tyrosine content is also species-dependent. Elastoidin, from Galeoscerdo cuveir (Tiger Shark) and another unidentified species, on treatment with formic acid yielded three fractions A,B and C. On the basis of analytical data it appears that specimens of elastoidin containing no (or little) tryptophan may yield fraction B through the solubilization of fraction A by formic acid. C fractions from two specimens of fibres were collagenous in nature. C fractions have been further purified in this study by charcoal treatment which removes a tyrosine-rich contaminant, to yield collagens with only approx. 2–4 residues of tyrosine per assumed mol. wt. of 360000. In the collagen from the unidentified species glucose and galactose were present in the ratio of 2:5; some glucosamine was also present.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.