I02-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Johan P.
Turkenburg
,
Elena
Blagova
,
Jitka
Waterman
,
Antonio
Ariza
,
Li
Ming
,
Sun
Tianqi
,
Carsten
Andersen
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[1221, 9948]
Open Access
Abstract: 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.
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Oct 2019
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I02-Macromolecular Crystallography
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Radka
Chaloupkova
,
Veronika
Liskova
,
Martin
Toul
,
Klara
Markova
,
Eva
Sebestova
,
Lenka
Hernychova
,
Martin
Marek
,
Gaspar P.
Pinto
,
Daniel
Pluskal
,
Jitka
Waterman
,
Zbyněk
Prokop
,
Jiří
Damborský
Diamond Proposal Number(s):
[4923]
Abstract: To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Crystal structure revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants, and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen-deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl-enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. In conclusion: a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at enzyme class-level; and ancestral reconstruction has a clear potential for obtaining multi-functional catalysts.
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Apr 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
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Sze Lei
Pang
,
Kok Lian
Ho
,
Jitka
Waterman
,
Robert Paul
Rambo
,
Aik-Hong
Teh
,
Indran
Mathavan
,
Gemma
Harris
,
Konstantinos
Beis
,
Yee-How
Say
,
Matta Sri
Anusha
,
Yang Yie
Sio
,
Fook Tim
Chew
,
Chyan Leong
Ng
Open Access
Abstract: Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.
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Mar 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Siti Nurulnabila
A. Rahaman
,
Jastina
Mat Yusop
,
Zeti-Azura
Mohamed-Hussein
,
Wan Mohd
Aizat
,
Kok Lian
Ho
,
Aik-Hong
Teh
,
Jitka
Waterman
,
Boon Keat
Tan
,
Hwei Ling
Tan
,
Adelicia Yongling
Li
,
Ee Sin
Chen
,
Chyan Leong
Ng
Diamond Proposal Number(s):
[5073, 11651]
Open Access
Abstract: 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.
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Sep 2018
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I04-Macromolecular Crystallography
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Koen
Beerens
,
Stanislav
Mazurenko
,
Antonin
Kunka
,
Sérgio M.
Marques
,
Niels
Hansen
,
Milos
Musil
,
Radka
Chaloupkova
,
Jitka
Waterman
,
Jan
Brezovsky
,
David
Bednar
,
Zbynek
Prokop
,
Jiri
Damborsky
Diamond Proposal Number(s):
[11175]
Abstract: 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 calculations. 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 computational demands. Altogether, our results strongly suggest that energetic
calculations should be complemented by a phylogenetic analysis in protein stabilization endeavors.
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Aug 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11175]
Abstract: 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.
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Feb 2017
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I02-Macromolecular Crystallography
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Abstract: 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%.
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Mar 2016
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Open Access
Abstract: Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, [beta] = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å3 Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
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Nov 2015
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Open Access
Abstract: With Diamond now firmly into its third phase of construction, the availability of new beamlines is allowing the industrial usage to thrive. The scope for industrial research and development is greater than ever before, allowing our clients to probe their systems of interest with greater resolution or under closer to realistic operating conditions. The establishment of the industrial liaison activity at Diamond, along with a description of our key market sectors, was described in a previous article [1]. The current status of industrial research at Diamond and an insight into some Europe-wide initiatives is given here.
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May 2014
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I04-Macromolecular Crystallography
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Antonio
Ariza
,
Olga V.
Moroz
,
Elena V.
Blagova
,
Johan P.
Turkenburg
,
Jitka
Waterman
,
Shirley M.
Roberts
,
Jesper
Vind
,
Carsten
Sjøholm
,
Søren F.
Lassen
,
Leonardo
De Maria
,
Vibe
Glitsoe
,
Lars K.
Skov
,
Keith S.
Wilson
Open Access
Abstract: Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.
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May 2013
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