Algorithms ; Crystallography, X-Ray ; Disulfides ; chemistry ; Models, Molecular ; Protein Conformation ; Proteins ; chemistry

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of the extracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichia coli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Catalytic Domain ; Crystallography, X-Ray ; Disulfides ; chemistry ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; chemistry ; metabolism ; Molecular Docking Simulation ; Molecular Sequence Data ; Mycobacterium tuberculosis ; metabolism ; Oxidation-Reduction ; Protein Disulfide-Isomerases ; chemistry ; metabolism ; Protein Folding ; Protein Structure, Tertiary ; Sequence Alignment ; Static Electricity

The molecular characterization and phylogenetic analysis of hemagglutinin (HA) genes of human influenza H3N2 viruses in Guangdong, China from 2007 to 2010 were studied in this study. By space-time sampling of strains, the HA genes of H3N2 strains from Guangdong were sequenced and searched from Internet, and then the variation and evolution of HA genes were conducted by Lasergene 7.1 and Mega 5.05 and evolutionary rates were analyzed by epidemiological data. The phylogenetic tree was established by alignment of 17 Guangdong strains and 26 global reference strains. Ks rates and Ka rates of HA genes were 2.06 x 10(-3)-2.23 x 10(-3) Nt/Year and 1.05 x 10(-3)-1.21 x 10(3) Nt/Year during 2007-2010, while the velocity of HA1 evolution of Ka was 3. 13 times than that of HA2 evolution. Compared with HA of vaccine strain A/Perth/16/2009, the genetic homologies of Guangdong strains in 2009 reached to 98.8%-99.7% and of Guangdong strains in 2010 reached to 98.0%-98.4%. There were some amino acid substitutions in five epitope regions of HA1 during 2007-2010, especially in B region (N160K) and D region (K174R/N); the K189E/N/Q and T228A in RBS (receptor-binding site) occurred in 2010 as two glycoproteins sites substituted impacted on the HA1 antigenicity. The antigenicity of epidemic H3N2 strains in 2010 was to some degree different that of the vaccine strain A/ Perth/16/2009. According to that there were variations of B and D epitopes and two sites of RBS and two glycoprotein in Guangdong H3N2 HA1 genes, WHO/ CDC should recommend new representative strains during 2011-2012 influenza seasons if H3N2 HA genes further evolve in the near future.
Amino Acid Substitution ; China ; Disulfides ; chemistry ; Epitopes ; genetics ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Mutation ; Phylogeny

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
Amino Acid Sequence ; Amino Acid Substitution ; Aspergillus oryzae ; enzymology ; Base Sequence ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

The recently identified interleukin-17 (IL-17) cytokines family, which comprises six members in mammals (IL-17A-F), plays essential roles in the host immunity against infectious diseases and chronic inflammatory diseases. The three-dimensional structures containing IL-17A or IL-17F have become available and revealed the unique structural features of IL-17s as well as their receptors. Molecular modeling in this review shows that IL-17s may adopt a "cysteine knot" fold commonly seen in nerve growth factor (NGF) and other neurotrophins. Further modeling analysis unmasks a signature interaction feature of the IL-17F/IL-17RA complex, where a small loop of IL-17RA slots into the deep groove of the interface of IL-17F homodimer. This is quite different from the interaction between the best known four-helix cytokines and their cognate receptors. On the other hand, structure of IL-17A and its monoclonal antibody (CAT-2200) shows that, albeit that the antigenic epitope of IL-17A resides outside of the IL-17A homodimer interface, its physical proximity to the receptor binding groove may explain that antibody blockage would be achieved by interfering with the ligand-receptor interaction. This review is to summarize the advance in understanding the structure and function of IL-17 family cytokines, focusing mainly on IL-17A, IL-17F and IL-17E, in the hope of gaining better knowledge of immunotherapeutic strategies against various inflammatory diseases.
Amino Acid Sequence ; Animals ; Conserved Sequence ; Cysteine ; Disulfides ; chemistry ; Humans ; Interleukin-17 ; chemistry ; metabolism ; Molecular Sequence Data ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptors, Interleukin ; metabolism

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange ; methods ; Disulfides ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Protein Refolding ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Tissue Plasminogen Activator ; analysis ; chemistry

We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50 degrees C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.
Amino Acid Substitution ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Mutagenesis, Site-Directed ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; Trichoderma ; enzymology ; genetics

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
ATP Binding Cassette Transporter, Sub-Family B ; immunology ; Antibodies, Bispecific ; biosynthesis ; chemistry ; genetics ; immunology ; Binding, Competitive ; CD3 Complex ; immunology ; Cell Line ; Disulfides ; chemistry ; Drug Stability ; Escherichia coli ; genetics ; metabolism ; Humans

To reveal the effects of disulfide bridges in rubella virus glycoprotein E1 on the membrane fusion activity, the recombinant plasmid pBSK-SPE2E1 and site-directed mutagenesis to mutate 11 cysteines individually in the ectodomain of E1 to remove a disulfide bridge from the wild-type E1 were constructed. All mutants and the wild-type plasmid were expressed on BHK-21 cell. Giemsa Staining was used to show the polykaryon formed in the transfected BHK-21 cells. The cell surface expression efficiency of the plasmids was assayed with fluorescence-activated cell sorter (FACS). Hemadsorption was performed to detect the receptor recognition activity of the recombinant plasmids. The results showed that all the 10 disulfide bridges in the ectodomain of E1 played an important role in the process of the membrane fusion. The removal of any disulfide bridge resulted in the loss of the fusion activity. The disulfide formed by the 5th and the 8th cysteine might be critical for the interaction of E1 and E2. While the disulfide bridges formed by the 3rd, the 4th, and the 13th might influence the membrane fusion activity of E1 directly.
Cell Membrane ; drug effects ; Cysteine ; chemistry ; Disulfides ; chemistry ; pharmacology ; Flow Cytometry ; Membrane Fusion ; drug effects ; Mutagenesis, Site-Directed ; Rubella virus ; chemistry ; Viral Envelope Proteins ; chemistry ; Viral Fusion Proteins ; Virus Internalization ; drug effects

We study the techniques of synthesis of disulfide bond-bearing cyclopeptides FIK. This experimentation with the material of Fmoc-aa use Solid-Phase synthesis after condensation by HBTU/HOBt/DIEA to synthesize linear peptide, then cyclopeptide was synthesized by creation of intramolecular disulfide bond by means of 12 oxidation of bis cysteine sulfhydryl of the linear peptide. The crude production was cleaved from the resin together with all protecting group and identified and separated by MALDI-MS and RP-HPLC. The peptide yield was 18%, after purification the purity was more than 97%. It was identified on MALDI-MS and Ellman reagent detection. This method is effective, simple, rapid and obtained good yield, and it's fit for the large-scale production.
Amino Acids ; chemistry ; Combinatorial Chemistry Techniques ; methods ; Disulfides ; chemistry ; Fluorenes ; chemistry ; Molecular Structure ; Peptides, Cyclic ; chemical synthesis ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization