[Show abstract][Hide abstract] ABSTRACT: β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α' and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α' and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2015; 71(Pt 2):132-5. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein.
Bioscience Biotechnology and Biochemistry 01/2015; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Geobacillus zalihae sp. nov., which produces a putative thermostable lipase, represents a novel species, with type strain T1. The characterisation of this intrinsically thermostable T1 lipase either physicochemically or structurally is an important task. The crystallisation of T1lipase in space was carried out using a High-Density Protein Crystal Growth (HDPCG) apparatus with the vapour diffusion method, and X-ray diffraction data were collected. The microgravity environment has improved the size and quality of the crystals as compared to earth grown crystal. The effect of microgravity on the crystallisation of T1 lipase was clearly evidenced by the finer atomic details at 1.35 Å resolution. Better electron densities were observed overall compared with the Earth-grown crystals, and comparison shows the subtle but distinct conformations around Na+ ion binding site stabilized via cation- interactions. This approach could be useful for solving structure and function of lipases towards exploiting its potentials to various industrial applications.
Protein and Peptide Letters 10/2014; · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.
Proceedings of the National Academy of Sciences 08/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3112 (or P3212), with unit-cell parameters a = b = 88.9, c = 221.2 Å, α = 90, β = 90, γ = 120°, indicating that a dimer was present in the asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2014; 70(Pt 6):738-741. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a new crystallization technique combining the solution stirring technique and crystallization at the interface between high-concentrated hydrogel and solution (the on-gel stirring technique). The solution stirring effect on the crystal quality of insulin was evaluated by an average difference between temperature factors determined by the slopes of the relative Wilson Plot. Higher-quality insulin crystals could be obtained under the stirred environment. The environmental stability of insulin crystals grown at the interface between hydrogel and solution for temperature change was tested. The dissolution point of a gel-interface crystal was 2 K higher than that of a solution-grown crystal. The quality of an insulin crystal grown by the on-gel stirring technique was finally evaluated and it was better than the solution-grown crystals with/without stirring and the gel-interface grown crystals. These results confirm that the on-gel stirring technique is a practical way to obtain high-quality crystals with improved environmental stability.
Japanese Journal of Applied Physics 05/2014; 53(6):065502. · 1.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12--S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.
[Show abstract][Hide abstract] ABSTRACT: SaFtsA and SaFtsZbind by comigration in non denaturing gel electrophoresis (View interaction)
SaFtsZ and SaFtsAbind by molecular sieving (View interaction)
SaFtsA and SaFtsAbind by x-ray crystallography (View interaction)
[Show abstract][Hide abstract] ABSTRACT: Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO2(-)) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate(-)bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η(1)-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen(-)bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. Based on these observations, the reaction mechanism of GtNIR through the η(1)-O coordination manner is proposed.
Journal of Biochemistry 11/2013; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.
Chemical Society Reviews 11/2013; · 30.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (1) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate. (2) The heat denaturation of SfTSA was completely reversible at pH 7.0, and the solubility of denatured SfTSA was higher than that of denatured EcTSA. The two-state transition of denaturation for SfTSA was highly cooperative, whereas the denaturation process of EcTSA was considerably more complex. (3) The global structure of SfTSA was quite similar to those of α subunits from other species. Relative to those other proteins, SfTSA exhibited an increase in cavity volume and a decrease in the number of ion pairs. SfTSA also lacks a hydrogen bond near loop B, related to catalytic function. These characteristics of SfTSA might provide the conformational flexibility required for catalytic activity at low temperatures.
Journal of Biochemistry 10/2013; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with β-γ-imidoadenosine 5'-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, β = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):895-898. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):884-887. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 Å. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2013; 69(Pt 7):719-22. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Agarose has been utilized in protein crystallization to control nucleation of protein crystals. It reduces convection, prevents crystal sedimentation, and increases tolerance to environmental perturbations, resulting in high-quality protein crystals. However, crystallographers have seldom used agarose hydrogel because it requires preincubating the crystallization solution at high temperatures where a high-temperature-sensitive protein may be inactivated or aggregated. To overcome this disadvantage, we used a thermoreversible gel polymer (TGP) made from synthetic polymer. TGP turns into hydrogel upon warming and liquefies upon cooling. This novel approach enabled us to prepare the crystallization solutions at low temperature (277–283 K) and to crystallize elastase, glucose isomerase, and lysozyme with TGP. We also found that TGP clearly increased the number of elastase, glucose isomerase, and lysozyme crystals. This approach will provide a wide variety of possibilities for protein crystallization in hydrogels.
[Show abstract][Hide abstract] ABSTRACT: The effect of gel–solution interfaces on the femtosecond laser-induced nucleation of proteins was investigated. Using hen egg white lysozyme, we found that the nucleation efficiency could be modulated by the distance between the laser focus and agarose gel surfaces (h) and the gel concentration (c). In particular, laser irradiation near the soft gel surface (h = 50 μm, c = 0.5%) could induce nucleation at very low supersaturation, where no nucleation could be induced in the bulk solutions even with femtosecond laser irradiation. Such enhancement of the nucleation was also confirmed for the membrane protein, acriflavine resistance protein B (AcrB), and an organic compound, N-(4-hydroxyphenyl)acetamide (paracetamol). To gain further insights into the role of gel–solution interfaces, we conducted fast imaging of cavitation bubbles, which are known to locally concentrate supersaturated solutions and thus act as a trigger for laser-induced nucleation. We found that the cavitation bubbles shrank asymmetrically near the soft gel surfaces and then finally collapsed toward the solution side. This is in contrast to the symmetric collapse in bulk solutions, which generates a large amount heat and chemical decomposition at the focus. In addition, cavitation bubbles generated near stiff gel surfaces (c ≥ 2%) caused an axial liquid jet toward the gel surfaces, which would disturb the formation of locally concentrated regions. These results suggest that gel–solution interfaces significantly modulate cavitation bubble dynamics and can be cues to achieve effective nucleation.
[Show abstract][Hide abstract] ABSTRACT: Protein crystals are required for X-ray crystallography to determine
three-dimensional structures of proteins at atomic resolution. The
conventional microscopy is currently used for observation and screening
of protein crystals. However, the three-dimensional imaging, which is
important for automated treatment of protein crystals, is generally
difficult by light microscopy. In addition, the protein crystals in the
media are frequently difficult to identify by conventional light
microscopy owing to the appearance of salt crystals or amorphous
materials. In this work, we successfully demonstrated micro-scale,
non-invasive, three-dimensional cross-sectional imaging of protein
crystals using ultrahigh resolution optical coherence tomography
(UHR-OCT). A low noise, Gaussian like, high power supercontinuum at
wavelength of 800 nm was used as the light source. The axial resolution
of 2 um in sample and the sensitivity of 95 dB were achieved. Since the
protein crystal has homogeneous nano-structure, the optical scattering
is negligibly small. Therefore, we used gel-inclusion technique to
enhance the intensity of scattered signals, and clear, sharp 3D
cross-sectional images of protein crystals were successfully observed.
As the gel concentration was increased, the OCT signal intensity was
increased. Using this method, the protein crystals surrounded by
substantial amount of precipitates could be visualized, which is
difficult by conventional light microscopy. The discrimination of
protein and salt crystals was also demonstrated by the OCT signal
intensity. The wavelength dependence of OCT imaging for protein crystal
was examined at wavelength of 800-1700 nm regions. It was confirmed that
the finest images were observed using 800 nm wavelength system.
Proceedings of SPIE - The International Society for Optical Engineering 01/2013; · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a spatially precise, soft microseeding method for the production of single protein crystals that are suitable for X-ray crystallographic studies. We used focused femtosecond laser pulses to produce, via multiphoton absorption processes, seed crystals from small regions (1 μm2) of crystals. Hen egg-white lysozyme seed crystals, produced in this manner, grew to be single crystals without any deterioration in their crystallinity. We also validated the technique using polycrystals for the membrane protein, acriflavine resistance protein B, for which single crystals are very difficult to obtain. In addition, we found that the shape of a tetragonal lysozyme crystal prepared from the seed could be controlled by altering the time interval between the initiation of crystallization and laser ablation. We also tried to comprehend the mechanism of femtosecond laser-induced microseeding. We visualized the ablated surfaces of the lysozyme crystals by atomic force microscopy and by laser confocal microscopy combined with differential interference microscopy. The results obtained in this study clearly demonstrate that femtosecond laser ablation of protein crystals is based on a photomechanical process, which ejects crystal fragments with little thermal damage. Femtosecond laser ablation is indeed very promising to produce high quality protein seed crystals from polycrystals or cracked crystals that are not suitable for X-ray diffraction studies.