NMR assignments of the intrinsically disordered K2 and YSK2 dehydrins

Dehydrins are proteins expressed by plants during various dehydrative stresses (drought, cold, and high salinity) to reduce cellular damage. These intrinsically disordered proteins are thought to function by binding to proteins to prevent denaturation, and to membranes to prevent leakage. Here, we report the ¹H, ¹⁵N, and ¹³C chemical shift assignments of the K₂ and YSK₂ dehydrins from Vitis riparia (wild grape). Our results show that the segmental nature of dehydrins allows for the assignments of the shorter dehydrins to be used in that of the longer forms.     

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6?×?His–TF–CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF–CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase–human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25?mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.     

Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity

Although eukaryotic Hsp90 has been studied extensively, the function of its bacterial homologue HtpG remains elusive. Here we report that 50S ribosomal protein L2 was found as an associated protein with His-tagged HtpG from Escherichia coli cultured in minimum medium at 45 °C. L2 specifically activated ATPase activity of HtpG, but other denatured proteins did not. The analysis using domain derivatives of HtpG and L2 showed that C-terminal domain of L2 and the middle to C-terminal domain of HtpG are important for interaction. At physiological salt concentration, L2 was denatured state and was recognized by HtpG as well as other chaperones, DnaK/DnaJ/GrpE and GroEL/GroES. The ATPase of HtpG at increasing concentration of L2 indicated that an L2 molecule bound to a dimer HtpG with apparent K_D of 0.3 μM at 100 mM KCl and 3.3 μM at 200 mM KCl.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Amine functional monodisperse microbeads via precipitation polymerization of N-vinyl formamide: immobilized laccase for benzidine based dyes degradation

Densely cross-linked poly(vinylamine) microbeads (∼2 μm) were prepared by precipitation copolymerization of N-vinyl formamide and ethylene glycoldimethacrylate in acetonitrile. The formamido groups of the microbeads were hydrolyzed into amino groups. Then, amino-functionalized microbeads were used for covalent immobilization of laccase via glutaraldehyde coupling. The average amount of immobilized enzyme was 18.7 mg/g microbeads. Kinetic parameters, V_max and K_m values were determined as 20.7 U/mg protein and 2.76 × 10⁻² mmol/L for free enzyme and 15.8 U/mg protein and 4.65 mmol/L for the immobilized laccase, respectively. The immobilized laccase was operated in a batch reactor for the degradation of two different benzidine based dyes (i.e., Direct Blue 1 and Direct Red 128). The laccase immobilized on the microbeads was very effective for removal of these dyes which interfere with the hormonal system.     

Targeting intrinsically disordered proteins in neurodegenerative and protein dysfunction diseases: another illustration of the D2 concept

Many biologically active proteins, which are usually called intrinsically disordered or natively unfolded proteins, lack stable tertiary and/or secondary structure under physiological conditions in vitro. Their functions complement the functional repertoire of ordered proteins, with intrinsically disordered proteins (IDPs) often being involved in regulation, signaling and control. Their amino acid sequences and compositions are very different from those of ordered proteins, making reliable identification of IDPs possible at the proteome level. IDPs are highly abundant in various human diseases, including neurodegeneration and other protein dysfunction maladies and, therefore, represent attractive novel drug targets. Some of the aspects of IDPs, as well as their roles in neurodegeneration and protein dysfunction diseases, are discussed in this article, together with the peculiarities of IDPs as potential drug targets.     

Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1

Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1_642-890-GST-NBD2_1286-1531 was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K_m = 2.17 mM, V_max = 12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.     

Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEKL

The fusion protein of enterokinase light chain, DsbA-rEK_L, was expressed mainly in the inclusion body in E. coli. The recombinant bacteria were fermented to high density, with high expression of the fusion protein. After being washed with 0.5 % Triton X-100 and 4 mol/L urea, the inclusion body was dissolved in 6 mol/L guanidine and 100 mmol/L DTT, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03 mg/mL of fusion protein until its final concentration reached 0.3 mg/mL. The refolded protein was autocleaved, and the active EK_L molecule was released after the addition of 2 mmol/L of CaCl₂. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEK_L was found to be above 95 %, with a high activity to cleave the recombinant reteplase fusion protein, Trx-rPA. The yield of purified rEK_L was more than 60 mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large scale in a way such as expressed in the form of fusion proteins.     

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755 kDa. The NH₂-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K_cat/K_m) towards natural, modified, and synthesis substrates with optimal activity at 60 °C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg²⁺, Ca²⁺ and Ba²⁺. The structural properties of AprB, like a higher ratio of R/(R + K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN′, AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.     

Molecular Cloning, Expression, and Purification of Nuclear Inclusion A Protease from Tobacco Vein Mottling Virus

The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The K_m and k_cat values were about 0.2 mM and 0.071 s^–1, respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.     

Performance and bacterial compositions of aged refuse reactors treating mature landfill leachate

Aged landfill leachates become more refractory over time and difficulty to treat. Recently, aged refuse bioreactors show great promise in treating leachates. In this study, aged refuse bioreactors were constructed to simulate landfill leachate degradation process. The characteristics of leachate were: COD_cr, ∼2200 mg/L; BOD₅, ∼280 mg/L; total nitrogen, ∼2030 mg/L; and ammonia, ∼1900 mg/L. Results showed that bioreactor could remove leachate pollutants effectively at hydraulic loading of 20 L/m³ d. The removal rate reduced when hydraulic loading doubled or temperature lowered. Effluent recirculation could alleviate the temperature effect. Combining aged refuse and slag biofilters could treat leachate more efficiently. Pyrosequencing analysis indicated that bacteria from Pseudomonas, Lysobacter, Bacillus and δ-proteobacter, Flexibacteraceae were more abundant in the samples. The Shannon index decreased at lower temperature, while evenness and equitability increased with recirculation. We suggest that filter medium and temperature may be the main factors for shaping bacterial community structure.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

A membrane-free baffled microbial fuel cell for cathodic reduction of Cu(II) with electricity generation

A membrane-free baffled microbial fuel cell (MFC) was developed to treat synthetic Cu(II) sulfate containing wastewater in cathode chamber and synthetic glucose-containing wastewater fed to anode chamber. Maximum power density of 314 mW/m³ with columbic efficiency of 5.3% was obtained using initial Cu²⁺ concentration of 6400 mg/L. Higher current density favored the cathodic reduction of Cu²⁺, and removal of Cu²⁺ by 70% was observed within 144 h using initial concentration of 500 mg/L. Powder X-ray diffraction (XRD) analysis indicated that the Cu²⁺ was reduced to Cu₂O or Cu₂O plus Cu which deposited on the cathode, and the deficient cathodic reducibility resulted in the formation of Cu₄(OH)₆SO₄ at high initial Cu²⁺ concentration (500-6400 mg/L). This study suggested a novel low-cost approach to remove and recover Cu(II) from Cu²⁺-containing wastewater using MFC-type reactor.     

Curcumin prevents Cr(VI)-induced renal oxidant damage by a mitochondrial pathway

We report the role of mitochondria in the protective effects of curcumin, a well-known direct and indirect antioxidant, against the renal oxidant damage induced by the hexavalent chromium [Cr(VI)] compound potassium dichromate (K₂Cr₂O₇) in rats. Curcumin was given daily by gavage using three different schemes: (1) complete treatment (100, 200, and 400 mg/kg bw 10 days before and 2 days after K₂Cr₂O₇ injection), (2) pretreatment (400 mg/kg bw for 10 days before K₂Cr₂O₇ injection), and (3) posttreatment (400 mg/kg bw 2 days after K₂Cr₂O₇ injection). Rats were sacrificed 48 h later after a single K₂Cr₂O₇ injection (15 mg/kg, sc) to evaluate renal and mitochondrial function and oxidant stress. Curcumin treatment (schemes 1 and 2) attenuated K₂Cr₂O₇-induced renal dysfunction, histological damage, oxidant stress, and the decrease in antioxidant enzyme activity both in kidney tissue and in mitochondria. Curcumin pretreatment attenuated K₂Cr₂O₇-induced mitochondrial dysfunction (alterations in oxygen consumption, ATP content, calcium retention, and mitochondrial membrane potential and decreased activity of complexes I, II, II-III, and V) but was unable to modify renal and mitochondrial Cr(VI) content or to chelate chromium. Curcumin posttreatment was unable to prevent K₂Cr₂O₇-induced renal dysfunction. In further experiments performed in curcumin (400 mg/kg)-pretreated rats it was found that this antioxidant accumulated in kidney and activated Nrf2 at the time when K₂Cr₂O₇ was injected, suggesting that both direct and indirect antioxidant effects are involved in the protective effects of curcumin. These findings suggest that the preservation of mitochondrial function plays a key role in the protective effects of curcumin pretreatment against K₂Cr₂O₇-induced renal oxidant damage.     

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 2³ + 4 were performed to evaluate the following parameters: molar mass of PEG (M_PEG), concentration of PEG (C_PEG), concentration of citrate buffer (C_Cit), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of M_PEG = 10,000 g/mol, C_PEG = 22 wt%, C_Cit = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 μmol Trolox equivalent/mg protein, for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 μmol Trolox/mg protein). Nevertheless, the IC₅₀ values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.     

Tolerance of a ruminant ciliate <Emphasis Type="Italic">Entodinium caudatum</Emphasis> against mercury,copper and chromium

The effects of three heavy metal cations, mercury (II), copper (II), and chromium (VI), on the growth of the rumen ciliate Entodinium caudatum in vitro culture was studied. The E. caudatum culture was challenged by HgCl₂, CuCl₂, and K₂Cr₂O₇ for a period of 4 days. The tested concentrations of mercury (II) and copper (II) were 1, 5, 10, 20, 50 mg/L and 2, 10, 20, 40 mg/L for chromium (VI) at single dose with either untreated or inhibited bacterial co-culture population. Effective metal concentrations required to reduce ciliate growth by 50% (EC₅₀) for mercury (II), copper (II), and chromium (VI) either with untreated or inhibited bacterial co-culture population after 24 h of metal application were 24, 20, and 21 or 15, 20, and 19 mg/L, respectively. After 4 days of metal application, corresponding EC₅₀ values for mercury (II), copper (II), and chromium (VI) were 16, 20, and 17 (with untreated bacterial population) or not determinable, 20, and 15 mg/L, respectively (with inhibited bacterial population). Increased sensitivity of E. caudatum to tested heavy metals with inhibited bacterial co-culture population indicate that the ciliate resistance to the heavy metal tested depends on detoxification abilities of rumen bacterial population.     

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a V_max of 12 µmol/min/mg protein and a K_m of 45 μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5–7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.     

Isolation,identification and characterization of a novel Rhodococcus sp. strain in biodegradation of tetrahydrofuran and its medium optimization using sequential statistics-based experimental designs

Statistics-based experimental designs were applied to optimize the culture conditions for tetrahydrofuran (THF) degradation by a newly isolated Rhodococcus sp. YYL that tolerates high THF concentrations. Single factor experiments were undertaken for determining the optimum range of each of four factors (initial pH and concentrations of K₂HPO₄ · 3H₂O, NH₄Cl and yeast extract) and these factors were subsequently optimized using the response surface methodology. The Plackett–Burman design was used to identify three trace elements (Mg²⁺, Zn²⁺and Fe²⁺) that significantly increased the THF degradation rate. The optimum conditions were found to be: 1.80 g/L NH₄Cl, 0.81 g/L K₂HPO₄ · 3H₂O, 0.06 g/L yeast extract, 0.40 g/L MgSO₄ · 7H₂O, 0.006 g/L ZnSO₄ · 7H₂O, 0.024 g/L FeSO₄ · 7H₂O, and an initial pH of 8.26. Under these optimized conditions, the maximum THF degradation rate increased to 137.60 mg THF h⁻¹ g dry weight in Rhodococcus sp. YYL, which was nearly five times of that by the previously described THF degrading Rhodococcus strain.     

Evolutionarily Conserved Network Properties of Intrinsically Disordered Proteins

BackgroundIntrinsically disordered proteins (IDPs) lack a stable tertiary structure in isolation. Remarkably, however, a substantial portion of IDPs undergo disorder-to-order transitions upon binding to their cognate partners. Structural flexibility and binding plasticity enable IDPs to interact with a broad range of partners. However, the broader network properties that could provide additional insights into the functional role of IDPs are not known.ResultsHere, we report the first comprehensive survey of network properties of IDP-induced sub-networks in multiple species from yeast to human. Our results show that IDPs exhibit greater-than-expected modularity and are connected to the rest of the protein interaction network (PIN) via proteins that exhibit the highest betweenness centrality and connect to fewer-than-expected IDP communities, suggesting that they form critical communication links from IDP modules to the rest of the PIN. Moreover, we found that IDPs are enriched at the top level of regulatory hierarchy.ConclusionOverall, our analyses reveal coherent and remarkably conserved IDP-centric network properties, namely, modularity in IDP-induced network and a layer of critical nodes connecting IDPs with the rest of the PIN.