scholarly journals The coiled-coil domain of glycosomal membrane-associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis

2020 ◽  
Vol 76 (10) ◽  
pp. 464-468
Author(s):  
Anil Kumar Shakya ◽  
J. Venkatesh Pratap
Keyword(s):  
Leishmania Donovani ◽  
Protein Import ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Supramolecular Assembly ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Monoclinic Space Group ◽  
Cell Parameters ◽  
Coiled Coil Domain

The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98 Å resolution were obtained from a condition consisting of 1.4 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62 Å, β = 94.68°. Structure determination and characterization are in progress.

scholarly journals Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) fromDermatophagoides farinae

2015 ◽  
Vol 71 (11) ◽  
pp. 1396-1400 ◽  
Author(s):  
Sze Lei Pang ◽  
Kok Lian Ho ◽  
Jitka Waterman ◽  
Aik-Hong Teh ◽  
Fook Tim Chew ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
High Sequence Identity ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Dermatophagoides farinaeis 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 anEscherichia coliexpression 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 MTris–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 theC-centered monoclinic space groupC2, with unit-cell parametersa= 123.46,b= 27.71,c= 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å3 Da−1suggests 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.

scholarly journals Molecular cloning, overexpression, purification and crystallographic analysis of a GH43 β-xylosidase fromBacillus licheniformis

2015 ◽  
Vol 71 (8) ◽  
pp. 962-965 ◽  
Author(s):  
José Alberto Diogo ◽  
Leticia Maria Zanphorlin ◽  
Hélia Harumi Sato ◽  
Mario Tyago Murakami ◽  
Roberto Ruller
Keyword(s):  
Paper Industry ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Metal Affinity ◽  
Exclusion Chromatography ◽  
Hydrolysis Of

β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacteriumBacillus licheniformis(BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed inEscherichia coliBL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space groupC2, with unit-cell parametersa= 152.82,b= 41.9,c= 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.

scholarly journals A putative siderophore-interacting protein from the marine bacteriumShewanella frigidimarinaNCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

2016 ◽  
Vol 72 (9) ◽  
pp. 667-671 ◽  
Author(s):  
Inês B. Trindade ◽  
Bruno M. Fonseca ◽  
Pedro M. Matias ◽  
Ricardo O. Louro ◽  
Elin Moe
Keyword(s):  
Iron Acquisition ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Interacting Protein ◽  
X Ray ◽  
Cell Parameters ◽  
Unit Structure ◽  
Shewanella Frigidimarina

Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacteriumShewanella frigidimarinaNCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space groupP21, with unit-cell parametersa= 48.04,b= 78.31,c= 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.

scholarly journals X-ray crystallographic studies of the middle part of the human synaptonemal complex protein 1 coiled-coil domain

2015 ◽  
Vol 71 (9) ◽  
pp. 1131-1134 ◽  
Author(s):  
Hyun Ho Park
Keyword(s):  
Synaptonemal Complex ◽  
Short Form ◽  
Complex Structure ◽  
Coiled Coil ◽  
Middle Part ◽  
X Ray ◽  
Cell Parameters ◽  
Coiled Coil Domain ◽  
Complex Protein ◽  
Synaptonemal Complex Protein

The synaptonemal complex is a meiosis-specific complex structure formed at the synapse of homologous chromosomes to hold them together during meiosis. Synaptonemal complex protein 1 (SYCP1) is one of the components of the syneptonemal complex. In this study, the short form of the coiled-coil domain of SYCP1 was overexpressed inEscherichia coliwith an engineered C-terminal His tag. The short form of the coiled-coil domain of SYCP1 was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 3.0 Å from a crystal belonging to space groupI4, with unit-cell parametersa= 41.95,b= 41.95,c= 318.78 Å. The asymmetric unit was estimated to contain two molecules.

scholarly journals Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Santhosh Gatreddi ◽  
Sayanna Are ◽  
Insaf Ahmed Qureshi
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Protozoan Parasite ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Gene Encoding ◽  
Cell Parameters ◽  
Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

scholarly journals Production, crystallization and preliminary crystallographic analysis ofAllochromatium vinosumthiosulfate dehydrogenase TsdA, an unusual acidophilicc-type cytochrome

2014 ◽  
Vol 70 (10) ◽  
pp. 1424-1427 ◽  
Author(s):  
José A. Brito ◽  
André Gutierres ◽  
Kevin Denkmann ◽  
Christiane Dahl ◽  
Margarida Archer
Keyword(s):  
Crystallographic Analysis ◽  
Sulfur Cycle ◽  
Monoclinic Space Group ◽  
Asymmetric Unit ◽  
Oxidative Condensation ◽  
Unit Cell Parameters ◽  
Allochromatium Vinosum ◽  
Data Set ◽  
Purple Sulfur Bacterium ◽  
Cell Parameters

The ability to perform the very simple oxidation of two molecules of thiosulfate to tetrathionate is widespread among prokaryotes. Despite the prevalent occurrence of tetrathionate formation and its well documented significance within the sulfur cycle, little is known about the enzymes that catalyze the oxidative condensation of two thiosulfate anions. To fill this gap, the thiosulfate dehydrogenase (TsdA) enzyme from the purple sulfur bacteriumAllochromatium vinosumwas recombinantly expressed inEscherichia coli, purified and crystallized, and a crystallographic data set was collected. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 79.2,b= 69.9,c= 57.9 Å, β = 129.3°, contained one monomer per asymmetric unit and diffracted to a resolution of 1.98 Å.

scholarly journals Lumenal Endosomal and Golgi-Retrieval Determinants Involved in pH-sensitive Targeting of an Early Golgi Protein

2001 ◽  
Vol 12 (10) ◽  
pp. 3152-3160 ◽  
Author(s):  
Collin Bachert ◽  
Tina H. Lee ◽  
Adam D. Linstedt
Keyword(s):  
Transmembrane Domain ◽  
Coiled Coil ◽  
Ph Sensitive ◽  
Plasma Membrane Protein ◽  
Golgi Transport ◽  
Coiled Coil Domain ◽  
Golgi Localization ◽  
Golgi Protein ◽  
Lumenal Domain ◽  
Chimeric Molecules

Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is acis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon treatment with agents that disrupt lumenal pH, and it undergoes endosome-to-Golgi retrieval upon drug removal. Analysis of chimeric molecules containing domains from GPP130 and the plasma membrane protein dipeptidylpeptidase IV indicated that GPP130 targeting information is contained entirely within its lumenal domain. Dissection of the lumenal domain indicated that a predicted coiled-coil stem domain adjacent to the transmembrane domain was both required and sufficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieval. Further dissection of this stem domain revealed two noncontiguous stretches that each conferred Golgi localization separated by a stretch that conferred endosomal targeting. Importantly, in the absence of the endosomal determinant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Because monensin blocks endosome-to-Golgi transport, the finding that the endosomal determinant confers monensin sensitivity suggests that the endosomal determinant causes GPP130 to traffic to endosomes from which it is normally retrieved. Thus, our observations identify Golgi and endosomal targeting determinants within a lumenal predicted coiled-coil domain that appear to act coordinately to mediate retrieval-based targeting of GPP130.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of the ArsM arsenic(III)S-adenosylmethionine methyltransferase

2010 ◽  
Vol 66 (9) ◽  
pp. 1050-1052 ◽  
Author(s):  
Kavitha Marapakala ◽  
A. Abdul Ajees ◽  
Jie Qin ◽  
Banumathi Sankaran ◽  
Barry P. Rosen
Keyword(s):  
Crystallographic Analysis ◽  
Hazardous Substances ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Priority List ◽  
X Ray ◽  
Cell Parameters ◽  
Environmental Toxin

Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III)S-adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic algaCyanidioschyzonsp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 84.85,b= 46.89,c= 100.35 Å, β = 114.25° and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76 Å.

scholarly journals Cryo-EM structures of human TMEM120A and TMEM120B

2021 ◽  
Author(s):  
Meng Ke ◽  
Yue Yu ◽  
Changjian Zhao ◽  
Shirong Lai ◽  
Qiang Su ◽  
...  
Keyword(s):  
Potential Role ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Expression System ◽  
Coiled Coil ◽  
Fatty Acid Elongase ◽  
Coiled Coil Domain ◽  
Induced Currents ◽  
The Difference

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

scholarly journals Modulation of the Leishmania donovani Peroxin 5 Quaternary Structure by Peroxisomal Targeting Signal 1 Ligands

2004 ◽  
Vol 24 (17) ◽  
pp. 7331-7344 ◽  
Author(s):  
Kleber P. Madrid ◽  
Gregory De Crescenzo ◽  
Shengwu Wang ◽  
Armando Jardim
Keyword(s):  
Leishmania Donovani ◽  
Quaternary Structure ◽  
Protein Translocation ◽  
Coiled Coil ◽  
Size Exclusion ◽  
Oligomeric State ◽  
Dissociation Kinetics ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

ABSTRACT The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.

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