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1.  Molecular analysis of hyperthermophilic endoglucanase Cel12B from Thermotoga maritima and the properties of its functional residues 
Background
Although many hyperthermophilic endoglucanases have been reported from archaea and bacteria, a complete survey and classification of all sequences in these species from disparate evolutionary groups, and the relationship between their molecular structures and functions are lacking. The completion of several high-quality gene or genome sequencing projects provided us with the unique opportunity to make a complete assessment and thorough comparative analysis of the hyperthermophilic endoglucanases encoded in archaea and bacteria.
Results
Structure alignment of the 19 hyperthermophilic endoglucanases from archaea and bacteria which grow above 80°C revealed that Gly30, Pro63, Pro83, Trp115, Glu131, Met133, Trp135, Trp175, Gly227 and Glu229 are conserved amino acid residues. In addition, the average percentage composition of residues cysteine and histidine of 19 endoglucanases is only 0.28 and 0.74 while it is high in thermophilic or mesophilic one. It can be inferred from the nodes that there is a close relationship among the 19 protein from hyperthermophilic bacteria and archaea based on phylogenetic analysis. Among these conserved amino acid residues, as far as Cel12B concerned, two Glu residues might be the catalytic nucleophile and proton donor, Gly30, Pro63, Pro83 and Gly227 residues might be necessary to the thermostability of protein, and Trp115, Met133, Trp135, Trp175 residues is related to the binding of substrate. Site-directed mutagenesis results reveal that Pro63 and Pro83 contribute to the thermostability of Cel12B and Met133 is confirmed to have role in enhancing the binding of substrate.
Conclusions
The conserved acids have been shown great importance to maintain the structure, thermostability, as well as the similarity of the enzymatic properties of those proteins. We have made clear the function of these conserved amino acid residues in Cel12B protein, which is helpful in analyzing other undetailed molecular structure and transforming them with site directed mutagenesis, as well as providing the theoretical basis for degrading cellulose from woody and herbaceous plants.
doi:10.1186/1472-6807-14-8
PMCID: PMC3936955  PMID: 24529187
Cellulose; Conserved amino acid residues; Endoglucanase; Phylogenetic analysis; Thermostability
2.  Accurate Mass Spectrometry Based Protein Quantification via Shared Peptides 
Journal of Computational Biology  2012;19(4):337-348.
Abstract
In mass spectrometry-based protein quantification, peptides that are shared across different protein sequences are often discarded as being uninformative with respect to each of the parent proteins. We investigate the use of shared peptides which are ubiquitous (∼50% of peptides) in mass spectrometric data-sets for accurate protein identification and quantification. Different from existing approaches, we show how shared peptides can help compute the relative amounts of the proteins that contain them. Also, proteins with no unique peptide in the sample can still be analyzed for relative abundance. Our article uses shared peptides in protein quantification and makes use of combinatorial optimization to reduce the error in relative abundance measurements. We describe the topological and numerical properties required for robust estimates, and use them to improve our estimates for ill-conditioned systems. Extensive simulations validate our approach even in the presence of experimental error. We apply our method to a model of Arabidopsis thaliana root knot nematode infection, and investigate the differential role of several protein family members in mediating host response to the pathogen. Supplementary Material is available at www.liebertonline.com/cmb.
doi:10.1089/cmb.2009.0267
PMCID: PMC3317402  PMID: 22414154
ITRAQ; linear programming; mass spectrometry optimization; protein quantification; shared peptides
3.  Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase 
BMC Biotechnology  2012;12:28.
Background
Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity.
Results
When expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50 °C and thermal in-activation half-life (t1/2) at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates.
Conclusions
Domain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.
doi:10.1186/1472-6750-12-28
PMCID: PMC3413519  PMID: 22676349
Xylanase; Glucanase; Domain-swapping; Fusing
4.  2,2′-(1,3,5,7-Tetra­oxo-1,2,3,5,6,7-hexa­hydro­pyrrolo[3,4-f]isoindole-2,6-di­yl)diacetic acid N,N-dimethyl­formamide disolvate 
The asymmetric unit of the title compound, C14H8N2O8·2C3H7NO or L·2DMF (DMF = N,N-dimethyl­formamide), contains one half of the centrosymmetric mol­ecule L and one solvent mol­ecule, which is disordered between two orientations in a 0.555 (4):0.445 (4) ratio. Inter­molecular O—H⋯O hydrogen bonds link one L and two DMF mol­ecules into a centrosymmetric hydrogen-bonded cluster. The crystal packing is further stabilized by weak inter­molecular C—H⋯O hydrogen bonds.
doi:10.1107/S1600536809035107
PMCID: PMC2970400  PMID: 21577863

Results 1-4 (4)