scholarly journals Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form

Crystals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 466
Author(s):  
Fiora Artusio ◽  
Albert Castellví ◽  
Roberto Pisano ◽  
José A. Gavira
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Proteinase K ◽  
Dimensional Structure ◽  
Nucleation Density ◽  
Agarose Gels ◽  
Protein Mass ◽  
Diffusion Controlled ◽  
Set Up ◽  
The Ideal

Agarose gels provide the ideal environment for studying the nucleation step of complex biomacromolecules under diffusion-controlled conditions. In the present paper, we characterized the influence of agarose on the nucleation of three model proteins, i.e., lysozyme, insulin, and proteinase K, as a function of the agarose concentration using a batch method set-up inside flat capillaries. By using this set-up, we were able to directly count the number of crystals in a given volume and correlate it with the amount of agarose and with the average crystal size. We also studied the crystallization behavior of proteinase K with free-interface diffusion so that batch conditions were achieved through slow diffusion of the precipitant. Thanks to the control over the protein mass transport imposed by the network, a previously unknown crystal form, P212121, was obtained, and the three-dimensional structure was determined at a 1.6 Å resolution. Overall, the versatility of agarose gels makes them ideal candidates for the preparation of microcrystalline suspensions of biopharmaceuticals with precise and reproducible crystal attributes or for the exploration of the existence of different polymorphs.

scholarly journals Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (12) ◽  
pp. 1675-1682 ◽  
Author(s):  
Roie Dann ◽  
Shifra Lansky ◽  
Noa Lavid ◽  
Arie Zehavi ◽  
Valery Belakhov ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Average Unit

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

First identification of small-molecule inhibitors of Pontin by combining virtual screening and enzymatic assay

2012 ◽  
Vol 443 (2) ◽  
pp. 549-559 ◽  
Author(s):  
Judith Elkaim ◽  
Michel Castroviejo ◽  
Driss Bennani ◽  
Said Taouji ◽  
Nathalie Allain ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Small Molecule ◽  
Tumour Cell ◽  
Small Molecule Inhibitors ◽  
Colorimetric Assay ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Atp Binding Site ◽  
Set Up

The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular activities) family, is overexpressed in several cancers and its silencing in vitro leads to tumour cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumours for which the therapeutic arsenal is rather limited. The three-dimensional structure of Pontin has been resolved previously, revealing a hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, DrugScore and Xscore, structure-based virtual screening of 2200 commercial molecules was conducted into the ATP-binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than 20 top-scoring compounds, resulting in the identification of four ligands presenting an inhibition constant in the micromolar concentration range. Three of them inhibited tumour cell proliferation. The association of virtual screening and experimental assay thus proved successful for the discovery of the first small-molecule inhibitors of Pontin.

Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin.

1984 ◽  
Vol 3 (6) ◽  
pp. 1311-1314 ◽  
Author(s):  
A. Pähler ◽  
A. Banerjee ◽  
J.K. Dattagupta ◽  
T. Fujiwara ◽  
K. Lindner ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Proteinase K ◽  
Dimensional Structure ◽  
Three Dimensional Structure

Crystallization

2015 ◽  
Author(s):  
Roger G. Harrison ◽  
Paul W. Todd ◽  
Scott R. Rudge ◽  
Demetri P. Petrides
Keyword(s):  
Large Scale ◽  
Three Dimensional ◽  
Crystal Form ◽  
Solid Particles ◽  
Dimensional Structure ◽  
Internal Quality ◽  
Small Scale ◽  
X Ray Diffraction ◽  
Production Scale ◽  
Homogeneous Phase

Crystallization is the process of producing crystals from a homogeneous phase. For biochemicals, the homogeneous phase from which crystals are obtained is always a solution. Crystallization is similar to precipitation in that solid particles are obtained from a solution. However, precipitates have poorly defined morphology, while in crystals the constituent molecules are arranged in three-dimensional arrays called space lattices. In comparison to crystallization, precipitation occurs at much higher levels of supersaturation and rates of nucleation but lower solubilities. These and other differences between crystallization and precipitation are highlighted in Table 9.1. Because of these differences and because the theory of crystallization that has been developed is different from that for precipitation, crystallization is considered separately from precipitation. Crystallization is capable of producing bioproducts at very high purity (say, 99.9%) and is considered to be both a polishing step and a purification step. Polishing refers to a process needed to put the bioproduct in its final form for use. For some bioproducts, such as antibiotics, this final form must be crystalline, and sometimes it is even necessary that a specific crystal form be obtained. In some instances, the purification that can be achieved by crystallization is so significant that other more expensive purification steps such as chromatography can be avoided. There are actually two very different applications of crystallization in biotechnology and bioproduct engineering: crystallization for polishing and purification, and crystallization for crystallography. In the latter case, the goal is a small number of crystals with good size (0.2–0.9 mm) and internal quality. Although it has become common to crystallize proteins for characterization of their three-dimensional structure by x-ray diffraction, this is performed only at small scale in the laboratory, and the knowledge about how to crystallize proteins at large scale in a production process is less developed. However, many antibiotics and other small biomolecules are routinely crystallized in production scale processes. This chapter is oriented toward the use of crystallization in processes that can be scaled up.

scholarly journals Multi-crystal data collection using synchrotron radiation as exemplified with low-symmetry crystals of Dps

2020 ◽  
Vol 76 (11) ◽  
pp. 568-576
Author(s):  
Vladislav Kovalenko ◽  
Alexander Popov ◽  
Gianluca Santoni ◽  
Natalia Loiko ◽  
Ksenia Tereshkina ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Crystal Form ◽  
Dimensional Structure ◽  
Crystal Data ◽  
Data Set ◽  
Average Size ◽  
Low Symmetry

Multi-crystal data collection using synchrotron radiation was successfully applied to determine the three-dimensional structure of a triclinic crystal form of Dps from Escherichia coli at 2.0 Å resolution. The final data set was obtained by combining 261 partial diffraction data sets measured from crystals with an average size of approximately 5 µm. The most important features of diffraction data measurement and processing for low-symmetry crystals are discussed.

Observation of Aneurobracon philippinensis (Hymenoptera: Braconidae) Immatures Shows How Koinobiont Offspring Flexibly Adjust Their Development to Host Growth

2019 ◽  
Vol 112 (5) ◽  
pp. 490-496 ◽  
Author(s):  
Haruka Aoyama ◽  
Issei Ohshima
Keyword(s):  
Developmental Stages ◽  
Early Stage ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
First Instar ◽  
Leaf Mining ◽  
Wild Host ◽  
Host Growth ◽  
In The Wild ◽  
The Ideal

AbstractKoinobionts are parasitoids that allow their hosts to grow after infection, and they finally kill their host individuals at parasitoid-specific host stages. Since fatal accidents of host organisms directly result in the deaths of parasitizing koinobionts, a longer parasitization period in vulnerable hosts is likely to increase the mortality of the koinobionts. However, for hosts inhabiting concealed environments in their later developmental stages, koinobionts should begin parasitization in early-stage hosts to make use of the grown hosts. A koinobiont parasitoid, Aneurobracon philippinensis (Muesebeck), mainly uses a leaf-mining moth, Acrocercops transecta Meyrick (Lepidoptera: Gracillariidae) as a host. Due to the three-dimensional structure of the mines constructed by later instars of A. transecta, females of A. philippinensis seldomly oviposit into later instar hosts, whereas feeding on final instar hosts is essential for A. philippinensis larvae. This implies that oviposition targets in the wild are shifted to early instars, though the final instar is the ideal target to shorten the parasitization period. The dissection of wild host larvae demonstrated that no eggs were observed in the final instar, supporting the above expectation. Laboratory parasitization experiments revealed that A. philippinensis eggs hatched approximately 80 h after oviposition, and hatched larvae stayed in the first instar until the host larvae completed making cocoons. These results suggest that the first-instar period of the parasitoid larvae functions as an adjusting period to synchronize the parasitoid and host developmental stages and that koinobiosis plays an important role in utilizing the final instar of A. transecta as a resource.

scholarly journals MnBa2(HPO4)2(H2PO4)2

2012 ◽  
Vol 68 (6) ◽  
pp. i49-i49 ◽  
Author(s):  
Wei Sun ◽  
Li-Zhi Sun ◽  
Teng-Teng Zhu ◽  
Biao-Chun Zhao ◽  
Jin-Xiao Mi
Keyword(s):  
Hydrothermal Synthesis ◽  
Hydrogen Bonds ◽  
Title Compound ◽  
Ionic Radius ◽  
Three Dimensional ◽  
Structure Type ◽  
Dimensional Structure ◽  
Coordination Polyhedra ◽  
Oh Groups ◽  
Set Up

Crystals of manganese(II) dibarium bis(hydrogenphosphate) bis(dihydrogenphosphate), MnBa2(HPO4)2(H2PO4)2, were obtained by hydrothermal synthesis. The title compound is isotypic with its CdII and CaII analogues. The structure is built up of an infinite {[Mn(HPO4)2(H2PO4)2]4−} n chain running along [100], which consists of alternate MnO6 octahedra and [PO4] tetrahedra, in which the centrosymmetric MnO6 octahedra share their four equatorial O-atom corners with tetrahedral [PO3(OH)] groups and their two axial apices with tetrahedral [PO2(OH)2] groups. These chains are held together by BaO9 coordination polyhedra, developing into a three-dimensional structure. The O—H...O hydrogen bonds additionally stabilize the structural set-up. Due to the ionic radius of Mn2+ being much smaller than those of Ca2+ and Cd2+, this may imply that their adopted structure type has a great tolerance for incorporating various ions and the exploitation of more diverse compounds in the future is encouraged.

Computer modelling of three-dimensional dynamics of fast reconnection

1991 ◽  
Vol 45 (2) ◽  
pp. 251-266 ◽  
Author(s):  
M. Ugai
Keyword(s):  
Current Sheet ◽  
Computer Modelling ◽  
Neutral Line ◽  
Current System ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Reconnection Process ◽  
Reconnection Region ◽  
Set Up ◽  
Fast Reconnection

Computer simulations are used to investigate the basic three-dimensional structure of the fast reconnection mechanism spontaneously developing from a long current sheet. It is shown that if three-dimensional effects (∂sol;∂z ≠ 0) are not so strong, a locally enhanced resistivity results in current-sheet thinning, and a fast reconnection process, involving switch-off shocks, is eventually set up in a region limited in the z direction. The fast reconnection process near the z = 0 plane becomes quasi-steady and two-dimensional (∂/∂z = 0), so that the well-known Petschek mechanism is fully applicable. Distinct plasma rarefaction occurs inside the fast reconnection region, so that fast-mode expansion may propagate in the z direction, and the resulting inflow velocity uz takes the magnetic field into the fast reconnection region and contracts the latter. The global current system undergoes drastic changes during the fast-reconnection development. The current flow, initially directed in the z direction, first converges towards the neutral line, and is then largely deflected away from this line in the inner reconnection region.

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