Resonance assignments of the 56 kDa chimeric avidin in the biotin-bound and free forms

Avidin is a homotetrameric ~56 kDa protein found in chicken egg white. Avidin’s ability to bind biotin with a very high affinity has widely been exploited in biotechnological applications. Protein engineering has further diversified avidin’s feasibility. ChiAVD(I117Y) is a product of rational protein engineering. It is a hyperthermostable synthetic hybrid of avidin and avidin-related protein 4 (AVR4). In this chimeric protein a 23-residue segment in avidin has been replaced with the corresponding sequence found in AVR4, and a point mutation at subunit interface 1–3 (and 2–4) has been introduced. Here we report the backbone and sidechain resonance assignments of the biotin-bound form of ChiAVD(I117Y) as well as the backbone resonance assignments of the free form.     

Relationship between the acid phosphatases of the Kurloff body and the major 30–35 kDa glycoproteins of the Kurloff cell

This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 μm diameter) periodic acid-Schiff-positive lysosomal inclusion body present in Kurloff cells (KC). KC AcPase were extracted, with similar yields, either with non-ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE-cellulose chromatography of such crude Dounce-extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I). The main protein content consisted of, as testified by SDS-PAGE analysis, major KC glycoproteins of 30–35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N-glycosylproteins among which the presence of α(2,6) sialoglycoproteins was previously established. Following electrophoresis on a 4–15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I. After Clostridium-derived sialidase digestion of peak I, the highly active bands observed at pH 3.5 – 5.2 disappeared. These zymographic patterns were similar to those obtained with crude extracts. After Concanavalin A (ConA)-Sepharose chromatography of peak I, the single ConA-bound glucosamine-labelled peak, eluted at 200 methyl-α-d-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activity. After SDS-PAGE analysis, the ConA-bound fraction appeared to correspond only to a single broad protein band in the 30–35 kDa zone. The Sambucus nigra agglutinin-reactivity of the latter band confirmed the α(2,6) sialylation pattern of KC AcPase. Moreover, their strong binding to ConA-Sepharose suggests that they could only correspond to the hybrid type of the N-glycosylproteins, since they could not correspond to the oligomannosidic type considering the absence of Galanthus nivalis agglutinin-positive structures.     

Backbone assignments of the 34 kDa ketopantoate reductase from E. coli

Ketopantoate reductase is an essential enzyme for pantothenate (vitamin B₅) synthesis and a potential antibiotic target. Here we report the ¹⁵N and ¹HN, ¹³C′, ¹³C^α and ¹³C^β chemical shift assignments of the 34 kDa ketopantoate reductase in its apo state.     

Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly ¹³C, ¹⁵N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.     

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.     

FT-ICR-MS characterization of intermediates in the biosynthesis of the α-methylbutyrate side chain of lovastatin by the 277 kDa polyketide synthase LovF

There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using (13)C-labeled malonyl-CoA to form α-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to the 4'-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high-resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the 'H' subunit of bacterial reaction centres?

Although the amino acid sequence of the 9 kDa (phospho)protein of chloroplasts has been determined, the function of this thylakoid membrane protein in photosynthetic electron transport and the reason for its physiological control remains unclear. In this paper, I briefly review the evidence which indicates that the phosphorylation of the 9 kDa protein results in a partial inhibition of photosynthetic oxygen evolution by increasing the stability of the semiquinone bound to QA the primary, plastoquinone-binding site of photosystem II (PS II). I propose that in its dephosphorylated state, the 9 kDa thylakoid membrane protein may serve PS II to ensure efficient photochemical charge separation by aiding the transfer of reducing equivalents out of the reaction centre to the attendant plastoquinone pool. This function is analogous to that proposed for the H-subunit of the reaction centre of photosynthetic eubacteria. Whether these two proteins have evolved from a common ancestral reaction centre protein is discussed in the light of a comparison of their amino acid sequences and predicted secondary structures.     

Comprehensive Enzymatic Analysis of the Cellulolytic System in Digestive Fluid of the Sea Hare Aplysia kurodai. Efficient Glucose Release from Sea Lettuce by Synergistic Action of 45 kDa Endoglucanase and 210 kDa ?-Glucosidase

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.     

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [¹⁵N-¹H]- and [¹³C-¹H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan ¹⁵N backbone positions and also resolved signals for ¹⁵N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [¹³C-¹H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.     

Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Extraribosomal functions associated with the C terminus of the 37/67?kDa laminin receptor are required for maintaining cell viability

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G₁ phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.     

In situ expression of 15 kDa interferon alpha responsive gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) and E12.0 to make a profile of the regulator genes for odontogenesis. Fifteen kDa interferon alpha responsive gene (Ifrg15) is one of several highly-expressed genes in the E12.0 mandible. The current study examined the precise expression patterns of Ifrg15 mRNA in the mouse mandibular first molar by in situ hybridization to evaluate the possible functional roles of this gene in odontogenesis. Ifrg15 mRNA was expressed in the epithelial and mesenchymal tissues of the mandible at E10.5 and E12.0. The Ifrg15 in situ signal was detected in the epithelial bud and the surrounding mesenchyme at E14.0, and was present in the enamel organ including the primary enamel knot, and in the underlying mesenchyme at E15.0. The in situ signal was restricted in the inner and outer enamel epithelia and the stratum intermedium at E16.0. The signal of Ifrg15 mRNA was further restricted to the inner enamel epithelium and the adjacent stratum intermedium at E17.0 and E18.0. Consequently, the expression of Ifrg15 mRNA was localized in the ameloblasts and odontoblasts at postnatal days 1.0 to 3.0. However, the in situ signal was markedly weaker than at the embryonic period. The expression of Ifrg15 mRNA was coincidently observed in various craniofacial organs as well as in the tooth germ. These results suggest that Ifrg15 is closely related to odontogenesis, especially the differentiation of the ameloblasts and odontoblasts, and to the morphogenesis of the craniofacial organs.     

The nitric oxide donor sodium nitroprusside requires the 18?kDa Translocator Protein to induce cell death

Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, β-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.     

Backbone assignments of the 26 kDa neuron-specific ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)

UCH-L1 is a member of the family of ubiquitin C-terminal hydrolases whose primary role is to hydrolyze small C-terminal adducts of ubiquitin to generate free ubiquitin monomers. Expression of UCH-L1 is highly specific to neurons and point mutations in this enzyme are associated with a hereditary form of Parkinson’s disease. Herein, we present the NMR backbone assignments of human UCH-L1, thus enabling future solution-state NMR spectroscopic studies on the structure and function of this important protein.     

甜菜坏死黄脉病毒75kDa通读蛋白基因54kDa片段的克隆及其序列分析

以甜菜坏死黄脉病毒(BNYVV)内蒙分离物的总RNA为模板,通过反转录和PcR扩增获得75kDa通读蛋白基因54kDa片段的目的片段。将其克隆到pGEM－7Zf(+)上并转化DH5α得到了含有完整s4kDa片段的重组子pGBW52。采用双脱氧终止法进行序列分析。结果表明内蒙分离物的54kDa片段全长为1509nt,与法国的F13分离物相比缺失了3个核苷酸。其核苷酸序列和由此推导的氨基酸序列的同源性分别为94．97％和96．42％．     

Dlmo基因编码一个32kDa的蛋白

为了获取研究Dlmo（果蝇LMO基因的简称）的功能信息,使用耦联网织红细胞体外翻译系统将Dlmo基因进行体外转录翻译得到32kDa的蛋白产物。该蛋白产物与抗人类LMO2蛋白的多抗抗体发生免疫沉淀,并得到了32kDa的阳性带。为了证实Dlmo基因在体外和体内翻译能得到同一大小蛋白,从2～4小时果蝇胚盘中分别抽提核和胞浆提取物进行Western印迹分析,免疫血清可以识别胞浆提取物中的32kDa蛋白,而在核提取物中未曾见到,证实体外和体内翻译产物相同。免疫组织化学的分析在0～4小时胚盘外周胞浆中有Dlmo基因的阳性染色信号,结果证实,Dlmo与人类LMO不同,其表达产物是一个胞浆蛋白。 Abstract：In order to gain some insight into a possible function of Dlmo gene,we used the TNT coupled reticulocyte lysate systems as an in vitro translation system to detect the Drosoplila protein. We found a 32kDa protein product.To demonstrate that the DLMO protein has the same size in vivo as in vitro we studied nuclear and cytoplasmic extracts from 2-4h embryos in Western blots. The immuno serum recognizes a 32kDa protein in the cytoplasm that is not present in the nucleus.The products were immune precipitated with the polyclonal anti-LMO2 antibody raised against the human protein and seen a positive band of 32kDa.Using immuno histochemical analysis was seen a positive staining in the basal cytoplasm of blastoderm embryos at 4 hours. This result confirms that the DLMO is a cytoplasmprotein, not like human LMO protein.