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microfluidic device
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SdsA, a sodium dodecyl sulfate hydrolase, fromPseudomonas aeruginosawas crystallized in three different crystal polymorphs and their three-dimensional structure was determined. The different polymorphs present different crystal packing... more
SdsA, a sodium dodecyl sulfate hydrolase, fromPseudomonas aeruginosawas crystallized in three different crystal polymorphs and their three-dimensional structure was determined. The different polymorphs present different crystal packing habits. One of the polymorphs suggests the existence of a tetramer, an oligomeric state not observed previously, while the crystal packing of the remaining two polymorphs obstructs the active site entrance but stabilizes flexible regions of the protein. Nonconventional crystallization methods that minimize convection, such as counterdiffusion in polyvinyl alcohol gel coupled with the influence of a 500 MHz (10.2 T) magnetic field, were necessary to isolate the poorest diffracting polymorph and increase its internal order to determine its structure by X-ray diffraction. The results obtained show the effectiveness of nonconventional crystallographic methods to isolate different crystal polymorphs.
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A new easy-to-use device has been designed and implemented for electric field-induced protein crystallization in a vapor-diffusion configuration. The device not only controls crystal nucleation by means of the electrical current, but also... more
A new easy-to-use device has been designed and implemented for electric field-induced protein crystallization in a vapor-diffusion configuration. The device not only controls crystal nucleation by means of the electrical current, but also favors crystal growth owing to its vapor-diffusion setup. Crystallization was conducted in the presence of an internal electric field and direct current. The proteins investigated were lysozyme, as model protein, and 2TEL–lysozyme (a synthetic protein consisting of two tandem alpha helix motifs connected to a lysozyme moiety). Lysozyme crystals that grew attached to the cathode were larger than those grown attached to the anode or in the absence of an electric current. On the other hand, crystals of 2TEL–lysozyme qualitatively showed a better X-ray diffraction pattern when grown in the presence of an electric current.
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This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some... more
This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to high-resolution X-ray crystallography are discussed.
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Research Interests: Chemistry, Crystallography, Electrochemistry, Atomic Force Microscopy, Medicine, and 13 moreMacromolecular X-Ray Crystallography, Biological Sciences, X Rays, Mathematical Sciences, Electron Transfer, Animals, Proteins, Electron Transport, High Pressure Liquid Chromatography, Cattle, Myocardium, Protein Conformation, and Biosensing Techniques
With the advent of the Structural Genomics initiatives, we have seen a host of new technologies emerge for automation of the tedious crystallization process. The latest robots and nanodispensers allow us to perform a larger number of... more
With the advent of the Structural Genomics initiatives, we have seen a host of new technologies emerge for automation of the tedious crystallization process. The latest robots and nanodispensers allow us to perform a larger number of crystallization screens faster with significantly less quantities of protein. There is a great temptation to harvest crystals directly from these small drops and immediately collect a data set---without any further improvement of the crystal quality. While this works well in some instances, there are many crystals that require further optimization before they are useful for structure determination. Optimization strategies will be presented for a variety of different proteins in the following areas: Protein preparation Crystallization--methods and conditions Cryocrystallography—cryosolvent selection, soaking tricks
Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a... more
Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg(2+) (LrdC-Mg(2+)) and in complex with inorganic pyrophosphate (PPi) (LrdC-Mg(2+)-PPi). Crystals of native LrdC-Mg(2+) diffracted to 2.50 Å resolution and belonged to the trigonal space group P3221, with unit-cell parameters a = b = 107.1, c = 89.2 Å. Crystals of the LrdC-Mg(2+)-PPi complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3221. Crystals of the LrdC-Mg(2+)-PPi complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space gro...
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Ferritin is an ubiquitous iron storage protein found in all kingdoms of life. Ferritin is essential for iron homeostasis and is involved in a wide range of physiological and pathological processes. Several structures of ferr itin in... more
Ferritin is an ubiquitous iron storage protein found in all kingdoms of life. Ferritin is essential for iron homeostasis and is involved in a wide range of physiological and pathological processes. Several structures of ferr itin in complex with small molecules and metal ions have been reported. Here we report the crystal structures of Horse Spleen Ferritin, in which the crystals were grown by employing a novel approach adopting the microbatch experiments performed in the presence and absence of electric field using a 2% agarose pellet of CdSO 4 . We observed that 1) these structures contain increased number of Cd ions as compared to the crystallization of same protein by others using different methods. 2) The externally applied electric field reduced the number of nucleation and with fewer nucleation the size of the crystals increased. 3) There is no significant conformational change observed among these structures. 4) Irrespective of the externally applied electric field, this aga...
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Micrometeorites (MMs) are small particles that account for most of the extraterrestrial material deposited on Earth. Synchrotron X-ray fluorescence and diffraction allowed for chemical and mineral characterization to distinguish MM from... more
Micrometeorites (MMs) are small particles that account for most of the extraterrestrial material deposited on Earth. Synchrotron X-ray fluorescence and diffraction allowed for chemical and mineral characterization to distinguish MM from atmospheric particulate. The relative components of iron, nickel, and other elements were considered in the identification of ferrous MM while high amounts of titanium were considered an indication that the particles were of atmospheric origin. Out of 100 samples collected by high school students and teachers, eight were taken to a synchrotron for analysis. Of those eight, three exhibited extraterrestrial compositions. X-ray absorption near-edge structure analysis revealed that the same three samples contained sulfide, the main sulfur form constituent in MM. X-ray microdiffraction analysis showed the presence of the minerals pentlandite and forsterite. Collectively, these results support the extraterrestrial nature of the three particles.
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There is an increasing appreciation for structural diversity of DNA that is of interest to both DNA nanotechnology and basic biology. Here, we have explored how DNA responds to torsional stress by building on a previously reported... more
There is an increasing appreciation for structural diversity of DNA that is of interest to both DNA nanotechnology and basic biology. Here, we have explored how DNA responds to torsional stress by building on a previously reported two-turn DNA tensegrity triangle and demonstrating that we could introduce an extra nucleotide pair (np) into the original sequence without affecting assembly and crystallization. The extra np imposes a significant torsional stress, which is accommodated by global changes throughout the B-DNA duplex and the DNA lattice. The work reveals a near-atomic structure of naked DNA under a torsional stress of approximately 14%, and thus provides an example of DNA distortions that occur without a requirement for either an external energy source or the free energy available from protein or drug binding.
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Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for... more
Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km ) was 1.7 mM with a kcat of 75 s(-1). Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) comple...
We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were... more
We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM min(-1) and 68.49 s(-1) respectively and 0.693 mM, 105.32 mM min(-1) and 89.57 s(-1), for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.
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ABSTRACT
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During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2to 2H2O at the trinuclear... more
During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures ofThermus thermophilusHB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holoTth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2reduction was found in a double conformation: Glu451a (∼7 Å from the TNC) and Glu451b (∼4.5 Å from the TNC). A positive peak of electron density above 3.5σ in anFo−Fcmap for Glu451a O∊2indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apoTth-MCO and in Hg-bound structures neither the positive peak nor do...