A single hydrophobic cleft in the <Emphasis Type="Italic">Escherichia coli</Emphasis> processivity clamp is sufficient to support cell viability and DNA damage-induced mutagenesis <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes. Interactions of the homodimeric Escherichia coli β clamp with its known partners involves multiple surfaces, including a hydrophobic cleft located near the C-terminus of each clamp protomer.     

Isolation and Partial Characterization of Antifungal Metabolites Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. IBA 33

Antifungal proteins produced by Bacillus sp. IBA 33 were purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. The two purified proteins inhibited the growth of Geotrichum candidum, the sour rot disease agent in lemon. The proteins were stable at 20 (3 months), 40, 60 and 100°C (30 min) and remained active after sterilization at 121°C for 15 min. Their hydrophobic nature was proved and when were developed with ninhydrin they did not show any free amino groups. The infrared spectrum showed vibrational modes corresponding to peptide, ester or ketone links and saturated CH links corresponding to long chain fatty acids. UV scan spectroscopy showed tyrosine and or tryptophan amino acids in their composition. The remarkable thermo-resistance of proteins may be a good feature to be used in the development of a new biocontrol method of Geotrichum candidum.     

Structural perturbation of α-crystallin and its chaperone-like activity

α-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of α-crystallin towards photo-induced aggregation of γ-crystallin, aggregation of insulin and on the refolding induced aggregation of β- and γ-crystallins. We observed that α-crystallin could prevent photo-aggregation of γ-crystallin and this chaperone-like activity of α-crystallin is enhanced several fold at temperatures above 30°C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that α-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or re-organized hydrophobic surfaces of α-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to α-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.     

N-terminus deletion affecting the preparation of soluble cyanobacterial glutaredoxin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutaredoxin has been implicated in maintenance of a normal cellular thiol/disufide ratio and the regeneration of oxidatively damaged proteins. In order to obtain more information about these important regulatory proteins in cyanobacteria, we have previously cloned and expressed the first cyanobacterial glutaredoxin gene ssr2061 in Escherichia coli. In this work, the second glutaredoxin gene slr1562 was studied. About 90% of Grx2061 coded by ssr2061 was produced in a soluble form while 90% of Grx1562 coded by slr1562 was found in inclusion bodies. To improve the production of soluble Grx1562, we constructed two mutants: Grx1562NC with cysteines in conserved site substituted by serines, and Grx1562M with N-terminus hydrophobic region deletion. Only the latter mutant was successfully expressed in soluble form with increased glutaredoxin activity and showed less sensitivity in oxidative stress. Spectroscopic analysis shows that the structure of Grx1562M with less hydrophobic nature could give more opportunity for protein solubility and could improve the substrate catalytic efficiency. These results suggest that hydrophobic N-terminus determines the insolubility of Grx1562 and may provide another strategy for increasing expression level of soluble heterologous proteins in E. coli. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 383–391.     

A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Ligand binding and self‐association cooperativity of β‐lactoglobulin

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self‐association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine β‐lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled‐equilibria revealed that dimerization exerts disparate effects on the ligand‐binding capacity of β‐lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of β‐lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution. Copyright © 2013 John Wiley & Sons, Ltd.     

High-yield production of hydrophobins RodA and RodB from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.     

Sequence analysis corresponding to the PPE and PE proteins in<Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and other genomes

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC 1551 strains of theMycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and β-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40–100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of theMethanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function inArchaea andBacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 β-strands. We suggest that proteins with AB repeats inMycobacterium tuberculosis and other genomes may be associated as surface antigens. TheM. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in theM. leprae genome compared to theM. tuberculosis genome.     

Improved gel electrophoresis matrix for hydrophobic protein separation and identification

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N′-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N′-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: −0.085) than in the classical gel (average GRAVY: −0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach.     

Extracellular production of lipoxygenase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on wheat protein

In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

Gene and protein structure of the mate recognition protein gene family in <Emphasis Type="Italic">Brachionus manjavacas</Emphasis> (Rotifera)

The mate recognition protein (MRP) gene is a member of a family of extracellular matrix protein genes, called MRP Motif Repeat (MMR) genes, with no known homologs. Two sets of MMR genes, designated MMR-A and MMR-B, were found in Brachionus manjavacas. MMR-B has previously been shown to encode the MRP in the Brachionus plicatilis species complex. MMR family genes share the same basic structure: a signal peptide sequence, followed by nearly identical 276 bp (MMR-A) or 261 bp (MMR-B) repeats, with a truncated final repeat. Each repeat of the predicted MMR-A and -B proteins is expected to have a secondary structure of 5 α-helices, ranging in length from 11 to 20 amino acids, separated by coils of 1–3 amino acids. Hydrophobic and hydrophilic amino acids are predicted to be partitioned to opposite sides of each α-helix, suggesting that MMR proteins are globular with a hydrophobic core. MMR-A and MMR-B proteins vary in their post-translational modifications, resulting in differences in size and charge, and likely causing differences in the physical properties of the proteins on the surface of the female, and their ability to be recognized by a receptor on a male rotifer. The identity of MMR gene repeats is theorized to be maintained by concerted evolution, through a process of unequal crossing over and/or gene conversion, with new mutations likely to be lost. Rarely, however, the same process of concerted evolution can rapidly spread a mutation across all of the repeats. When a mutation results in conformational changes in the protein detectable by males, it could lead to reproductive isolation and thereby to speciation. Thus, changes in MRP could be a driving force in the high degree of species diversity seen within the B. plicatilis cryptic species complex.     

膜蛋白晶体的生长及其进展

生物膜中与脂双层结合的蛋白质称为膜蛋白.由于它们具有很大的疏水表面以及既亲水又疏水的两性特点致使其纯化与结晶都十分困难.在膜蛋白晶体生长系统中引入小分子去污剂与小的两性分子获得突破性进展.迄今为止,结晶出来的膜蛋白为数不多.其中只有光合细菌绿色红假单胞菌及球型红假单胞菌的反应中心得到3分辨率的晶体结构与解析.一系列膜蛋白形成二维晶体,可用电子显微镜与像重构技术获得三维结构信息.     

Investigation of hydrophobic moment and hydrophobicity properties for transmembrane <Emphasis Type="Italic">α</Emphasis>-helices

Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane α-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment (<μH>) and the corresponding mean hydrophobicity (<H>). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, θ, fixed at 100°.     

MPRAP: An accessibility predictor for <Emphasis Type="Italic">a</Emphasis>-helical transmem-brane proteins that performs well inside and outside the membrane

Background  

In water-soluble proteins it is energetically favorable to bury hydrophobic residues and to expose polar and charged residues. In contrast to water soluble proteins, transmembrane proteins face three distinct environments; a hydrophobic lipid environment inside the membrane, a hydrophilic water environment outside the membrane and an interface region rich in phospholipid head-groups. Therefore, it is energetically favorable for transmembrane proteins to expose different types of residues in the different regions.     

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca²⁺ binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.     

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

Mutational analyses of Cry protein block7 polypeptides that facilitate the formation of protein inclusion in <Emphasis Type="Italic">Escherichia coli</Emphasis>

4AaCter is the polypeptide from the C-terminal extension of mosquitocidal Cry4Aa toxin, and facilitates formation of protein inclusion in Escherichia coli. It has been demonstrated that the use of 4AaCter as a peptide tag results in the efficient production of heterologous protein in E. coli. It has also been demonstrated that proteins are integrated, without losing their biological activities, into the protein inclusions. Although the mechanism to form protein inclusions in E. coli is unclear, highly conserved block7 sequence in 4AaCter is thought to be one of the functional factors. In this study, to analyze the ability of block7 to form protein inclusion, synthetic genes encoding the block7 polypeptide from selected 15 Cry proteins were constructed and expressed to produce glutathione S-transferase fusions in E. coli. Unexpectedly, only three of them (Cry5Ba, Cry32Aa, and Cry48Aa) formed protein inclusion as efficiently as that of Cry4Aa (>90% efficiency). The efficiencies in forming the protein inclusion were ranging from 39% to 66% for most of the tested block7s, and almost no protein inclusion was observed in Cry47Aa block7. This suggested that the ability of block7 to form the protein inclusion may vary with the type of Cry protein or the amino acid sequences. Mutational analyses revealed that substitution of the hydrophobic amino acids in block7 significantly affected the formation of protein inclusion, suggesting some important roles of these hydrophobic amino acid residues. Present results will contribute to develop a compact peptide tag based on block7 which forms the protein inclusion efficiently.     

Comparative analysis of vertebrate <Emphasis Type="Italic">PEPT1</Emphasis> and <Emphasis Type="Italic">PEPT2</Emphasis> genes

The plasma membrane transport proteins belong to SoLute Carrier 15 (SLC15) family and two members of this family have been characterized extensively in higher vertebrates, namely PEPT1 and PEPT2. Despite many efforts have made to define a pharmacophore model for efficient binding and transporting of substrates, there is not a comprehensive study performed to elucidate the evolutionary mechanisms among the SLC15 family members and to statistically evaluate sequence conservation and functional divergence between members. In this study, we compared and contrasted the rates and patterns of molecular evolution of 2 PEPT genes. Phylogenetic tree assembly with all available vertebrate PEPTs suggests that the PEPTs originated by duplications and diverged from a common protein at the base of the eukaryotic tree. Topological structure demonstrates both members share the similar hydrophobic domains (TMDs), which have been constrained by purifying selection. Although both genes show qualitatively similar patterns, their rates of evolution differ significantly due to an increased rate of synonymous substitutions in the structural domains in one copy, suggesting substantial differences in functional constraint on each gene. Site-specific profiles were established by posterior probability analysis revealing significantly divergent regions mainly locate at the hydrophobic region between predicted transmembrane domains 9 and 10 of the proteins. Thus, these results provide the evidence that several amino acid residues with reduced selective constraints are largely responsible for functional divergence between the paralogous PEPTs. These findings may provide a starting point for further experimental verifications.