Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å3 Da−1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%

Structural and functional characterisation of three novel fungal amylases with enhanced stability and pH tolerance

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization

Evolutionary analysis as a powerful complement to energy calculations for protein stabilization

Stability is one of the most important characteristics of proteins employed as biocatalysts, biotherapeutics, and biomaterials, and the role of computational approaches in modifying protein stability is rapidly expanding. We have recently identified stabilizing mutations in haloalkane dehalogenase DhaA using phylogenetic analysis but were not able to reproduce the effects of these mutations using force-field calculations. Here we tested four different hypotheses to explain the molecular basis of stabilization using structural, biochemical, biophysical, and computational analyses. We demonstrate that stabilization of DhaA by the mutations identified using the phylogenetic analysis is driven by both entropy and enthalpy contributions, in contrast to primarily enthalpy-driven stabilization by mutations designed by the force-field Comprehensive bioinformatics analysis revealed that more than half (53%) of 1 099 evolution-based stabilizing mutations would be evaluated as destabilizing by force-field calculations. Thermodynamic integration considers both folded and unfolded states and can describe the entropic component of stabilization, yet it is not suitable for predictive purposes due to its high computational demands. Altogether, our results strongly suggest that energetic calculations should be complemented by a phylogenetic analysis in protein-stabilization endeavors

Crystal structure and epitope analysis of house dust mite allergen Der f 21

10.1038/s41598-019-40879-xSCIENTIFIC REPORTS91complete

Crystal structure and functional analysis of human C1ORF123

Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast Schizosaccharomyces pombe (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family

Acknowledgements: The authors would like to acknowledge Universiti Kebangsaan Malaysia for financial support through the GUP-2017-070, DIP-2012-13 and GGPM-2012-069 grants. This research is also supported by the Ministry of Higher Education, Malaysia through the ERGS/1/2011/STG/UKM/01/15 grant. We thank Diamond Light Source, UK for synchrotron beam time (I02 and I03) and we acknowledge Juan Sanchez-Weatherby and Thomas Sorensen for assistance at station I02 beamline during data collection. We thank Malaysia Genome and Vaccine Institute, National Institute of Biotechnology Malaysia for the in-house X-ray diffractometer facility. We thank Teo Chee How for useful discussion in phylogenetic analysis. We thank Pravin Kumran for technical support in protein purification. We also thank David W. Rice, Patrick J. Baker and Jon R. Sayers for insightful comments.Funder: Ministry of Higher Education, Malaysia for ERGS/1/2011/STG/UKM/01/15Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family

Abstract Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family.

Acknowledgements: The authors would like to acknowledge Universiti Kebangsaan Malaysia for financial support through the GUP-2017-070, DIP-2012-13 and GGPM-2012-069 grants. This research is also supported by the Ministry of Higher Education, Malaysia through the ERGS/1/2011/STG/UKM/01/15 grant. We thank Diamond Light Source, UK for synchrotron beam time (I02 and I03) and we acknowledge Juan Sanchez-Weatherby and Thomas Sorensen for assistance at station I02 beamline during data collection. We thank Malaysia Genome and Vaccine Institute, National Institute of Biotechnology Malaysia for the in-house X-ray diffractometer facility. We thank Teo Chee How for useful discussion in phylogenetic analysis. We thank Pravin Kumran for technical support in protein purification. We also thank David W. Rice, Patrick J. Baker and Jon R. Sayers for insightful comments.Funder: Ministry of Higher Education, Malaysia for ERGS/1/2011/STG/UKM/01/15Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2