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1.
Audiol Neurootol ; 28(1): 63-74, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36075191

RESUMO

INTRODUCTION: The aim of this study was to better understand the onset time and factors associated with cochlear obliteration following translabyrinthine approach (TLA) surgery for large cerebellopontine angle tumors. METHODS: This retrospective cohort study included 117 patients with large cerebellopontine angle tumor (tumor diameter >2 cm) treated by TLA surgery from June 2011 to March 2019 in a single tertiary referral center. The Kaplan-Meier method with log-rank test was used to estimate cochlear patency survival and the association between survival and covariates, and the Cox proportional hazards regression analysis was used to identify possible factors associated with cochlear obliteration. RESULTS: Of the 117 patients included in our analysis, the median follow-up was 24.8 months. There were 30 (25.6%) patients in the cochlear obliteration group, and 87 (74.4%) in the patent cochlear group. Various degrees of cochlear obliteration was found in 25.6% patients in final MRI scan, comprised of 50% grade I, 30% grade II, and 20% grade III. Cochlear patency survival curves showed 94.0% at 3 months, 73.0% at 18 months, which plateaued after 20 months with a survival rate of 71.6%. In the multivariate Cox proportional hazards model, patients presented with postoperative hyperintense T1W cochlear signal had poorer cochlear patency survival compared to isointense T1W (HR = 4.15). Similarly, postoperative deteriorated facial function (HR = 4.52) and full IAC involvement of tumor (HR = 2.33) demonstrated a higher risks of cochlear obliteration after TLA surgery. CONCLUSION: The 2-year estimated cochlear patency rate was 71.6% in patients that received TLA. Cochlear obliteration can develop as early as 3 months post-surgery, with no new obliteration 20 months after the surgery and half of these patients got severe obliteration. Three factors associated with cochlear obliteration were identified including full IAC involvement of tumor, postoperative facial function deterioration, and postoperative hyperintense T1W cochlear signal.


Assuntos
Implante Coclear , Neuroma Acústico , Humanos , Neuroma Acústico/cirurgia , Estudos Retrospectivos , Cóclea/cirurgia , Implante Coclear/métodos , Imageamento por Ressonância Magnética/métodos
2.
Int J Mol Sci ; 23(16)2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-36012609

RESUMO

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.


Assuntos
Cristalinas , alfa-Cristalinas , Animais , Dicroísmo Circular , Cristalinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Resposta ao Choque Térmico , Espalhamento a Baixo Ângulo , Difração de Raios X
3.
Opt Lett ; 46(23): 5990-5993, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34851941

RESUMO

We report generation of a visible and near-infrared supercontinuum from a high-gain, ultra-broadband, and mirrorless Raman oscillator in a monolithic KTP crystal. The plane transverse to the pump axis resonates and traps off-axis Stokes waves and their frequency-upconverted components bouncing between two crystal surfaces via total internal reflection. The Raman gain is maximized with the Stokes polarization perpendicular to the plane of reflections. When pumped by a Q-switched Nd:YAG laser, the monolithic oscillator generates quasi-mode-locked Stokes pulses with octave-spanning spectral groups across the visible and near-infrared spectra between 540 and 1800 nm.

4.
Opt Lett ; 45(23): 6430-6433, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33258829

RESUMO

A laser is meant to emit coherent radiation at a particular wavelength. Here, we demonstrate a laser that is prohibited to emit at a particular wavelength, called a dark line in the emission background of its gain spectrum. Specifically, we installed a 150 µm thick etalon mirror on an ytterbium-doped fiber laser. The laser suffers from 100% loss at the resonance of the etalon and generates a dark line in its emission spectrum. The interplay among the etalon resonance, homogenous gain broadening, and gain competition allows wavelength tuning and multiple-color emission from the laser.

5.
Opt Lett ; 42(23): 4897-4900, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29216138

RESUMO

Lithium niobate is the most popular material for terahertz wave generation via stimulated polariton scattering (SPS), previously known to have a gain peak near 2 THz. Here we report the discovery of another phase-matched gain peak near 4 THz in lithium niobate, which greatly extends the useful gain spectrum of lithium niobate. Despite the relatively high 4 THz absorption in lithium niobate, the 4 THz SPS becomes dominant over the 2 THz one in an intensely pumped short lithium niobate crystal due to less diffraction-induced absorption and mode-area mismatch. We also demonstrate a signal-seeded OTPO that generates 1.4 nJ at 4.2 THz from lithium niobate with 17.5 mJ pump energy.

6.
Opt Express ; 24(11): 12043-54, 2016 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-27410125

RESUMO

We report an Nd:YAG laser pumped by light emission diodes (LEDs) at 750 nm. With 1% output coupling from a linear cavity containing a 2-cm long Nd:YAG crystal, the laser generated 37.5 µJ pulse energy at 1064 nm with M2 = 1.1 when pumped by 2.73-mJ LED energy in a 1-ms pulse at a 10 Hz rate. The measured optical and slope efficiencies for this linear-cavity laser are 1.36, and 9%, respectively. With 1 and 5% output couplings from a Z-cavity containing the same laser crystal, the lasers generated 346 and 288 µJ pulse energy with an optical efficiency of 3.4 and 2.8% and slope efficiency of 6.6 and 14%, respectively, for the same 1-ms pump pulse repeating at a 10 Hz rate. At the highest output from the Z-cavity, the measured M2 for the beam is 3.6.

7.
Opt Express ; 24(23): 25964-25973, 2016 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-27857335

RESUMO

We report superior terahertz parametric generation from potassium titanyl phosphate (KTP) over congruent-grown lithium niobate (CLN) and lithium tantalate (CLT) in terms of parametric gain and laser damage resistance. Under the same pump and crystal configurations, the signal emerged first from KTP, 5% Mg-doped CLN, CLN, and then finally from CLT. The signal growth rate in KTP was comparable to that in 5%-Mg-doped CLN, but the signal power from KTP reached a much higher value after all the other crystals were damaged by the pump laser. We further demonstrate seeded terahertz parametric amplification in an edge-cut KTP at 5.74 THz. The THz parametric amplifier (TPA) employs a 17-mm long KTP gain crystal, pumped by a passively Q-switched pump laser at 1064 nm and seeded by a continuous-wave diode laser tuned to the signal wavelength at 1086.2 nm. With 5.8-mJ energy in a 520-ps pump pulse and 100-mW seed signal power, we measured 5-W peak-power THz output from the KTP TPA with 22% pump depletion. In comparison, we measured no detectable THz output power from a similar edge-cut CLN TPA under the same pump power, detection scheme, and crystal configuration, when tuning the seed laser wavelength to 1072.2 nm and attempting to generate a radiation at 2.1 THz.

8.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 9): 2331-43, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25195747

RESUMO

Optimization of the initial phasing has been a decisive factor in the success of the subsequent electron-density modification, model building and structure determination of biological macromolecules using the single-wavelength anomalous dispersion (SAD) method. Two possible phase solutions (φ1 and φ2) generated from two symmetric phase triangles in the Harker construction for the SAD method cause the well known phase ambiguity. A novel direct phase-selection method utilizing the θ(DS) list as a criterion to select optimized phases φ(am) from φ1 or φ2 of a subset of reflections with a high percentage of correct phases to replace the corresponding initial SAD phases φ(SAD) has been developed. Based on this work, reflections with an angle θ(DS) in the range 35-145° are selected for an optimized improvement, where θ(DS) is the angle between the initial phase φ(SAD) and a preliminary density-modification (DM) phase φ(DM)(NHL). The results show that utilizing the additional direct phase-selection step prior to simple solvent flattening without phase combination using existing DM programs, such as RESOLVE or DM from CCP4, significantly improves the final phases in terms of increased correlation coefficients of electron-density maps and diminished mean phase errors. With the improved phases and density maps from the direct phase-selection method, the completeness of residues of protein molecules built with main chains and side chains is enhanced for efficient structure determination.


Assuntos
Elétrons , Estrutura Molecular , Cristalografia por Raios X
9.
Opt Lett ; 39(16): 4792-5, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25121876

RESUMO

We report simultaneous laser pulse shortening and wavelength conversion based on spectral-temporal correlation in high-gain optical parametric generation (OPG). By spectrally filtering the off-peak signal energy, we shortened a 560 ps pump pulse at 1064 nm to an 80 ps signal pulse at 1.5 µm from a 45 mm long PPLN optical parametric generator with 60 µJ pump energy from a passively Q-switched Nd:YAG laser. Using the same technique, we further demonstrated a 3.6 time shortened laser pulse at 1072 nm from noncollinearly phase matched OPG in a 44 mm long lithium niobate crystal with 3 mJ amplified pump energy from the same Nd:YAG laser.

10.
JACS Au ; 4(6): 2130-2150, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38938812

RESUMO

Monoclonal antibodies (mAbs) have gradually dominated the drug markets for various diseases. Improvement of the therapeutic activities of mAbs has become a critical issue in the pharmaceutical industry. A novel endo-ß-N-acetylglucosaminidase, EndoSz, from Streptococcus equisubsp. zooepidemicus Sz105 is discovered and applied to enhance the activities of mAbs. Our studies demonstrate that the mutant EndoSz-D234M possesses an excellent transglycosylation activity to generate diverse glycoconjugates on mAbs. We prove that EndoSz-D234M can be applied to various marketed therapeutic antibodies and those in development for antibody remodeling. The remodeled homogeneous antibodies (mAb-G2S2) produced by EndoSz-D234M increase the relative ADCC activities by 3-26-fold. We further report the high-resolution crystal structures of EndoSz-D234M in the apo-form at 2.15 Å and the complex form with a bound G2S2-oxazoline intermediate at 2.25 Å. A novel pH-jump method was utilized to obtain the complex structure with a high resolution. The detailed interactions of EndoSz-D234M and the carried G2S2-oxazoline are hence delineated. The oxazoline sits in a hole, named the oxa-hole, which stabilizes the G2S2-oxazoline in transit and catalyzes the further transglycosylation reaction while targeting Asn-GlcNAc (+1) of Fc. In the oxa-hole, the H-bonding network involved with oxazoline dominates the transglycosylation activity. A mobile loop2 (a.a. 152-159) of EndoSz-D234M reshapes the binding grooves for the accommodation of G2S2-oxazoline upon binding, at which Trp154 forms a hydrogen bond with Man (-2). The long loop4 (a.a. 236-248) followed by helix3 is capable of dominating the substrate selectivity of EndoSz-D234M. In addition, the stepwise transglycosylation behavior of EndoSz-D234M is elucidated. Based on the high-resolution structures of the apo-form and the bound form with G2S2-oxazoline as well as a systematic mutagenesis study of the relative transglycosylation activity, the transglycosylation mechanism of EndoSz-D234M is revealed.

11.
Opt Express ; 21(2): 2452-62, 2013 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-23389226

RESUMO

Optical parametric mixing is a popular scheme to generate an idler wave at THz frequencies, although the THz wave is often absorbing in the nonlinear optical material. It is widely suggested that the useful material length for co-directional parametric mixing with strong THz-wave absorption is comparable to the THz-wave absorption length in the material. Here we show that, even in the limit of the absorption loss exceeding parametric gain, the THz idler wave can grows monotonically from optical parametric amplification over a much longer distance in a nonlinear optical material until pump depletion. The coherent production of the non-absorbing signal wave can assist the growth of the highly absorbing idler wave. We also show that, for the case of an equal input pump and signal in difference frequency generation, the quick saturation of the THz idler wave predicted from a much simplified and yet popular plane-wave model fails when fast diffraction of the THz wave from the co-propagating optical mixing waves is considered.


Assuntos
Amplificadores Eletrônicos , Radiação Terahertz , Absorção , Transferência de Energia , Desenho de Equipamento , Análise de Falha de Equipamento
12.
Opt Lett ; 38(11): 1863-5, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23722770

RESUMO

We report broadband polarized supercontinuum generated from a doubly resonant parametric oscillator with a lithium triborate gain crystal installed in a noncritical phase-matching condition. The parametric oscillator is pumped by a Q-switched frequency-doubled Nd:YAG laser at a repetition rate of 20 Hz, producing a polarized spectrum with a width more than 324 THz or a wavelength range between 0.6 and 1.7 µm. The laser output is further extended to the blue part of spectrum via temperature-tuned second-harmonic generation in the same crystal.

13.
Acta Crystallogr D Struct Biol ; 79(Pt 2): 154-167, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36762861

RESUMO

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline α-galactosidase. The structure of AtAkαGal3 comprises three domains: an N-terminal domain with 13 antiparallel ß-strands, a catalytic domain with an (α/ß)8-barrel fold and a C-terminal domain composed of ß-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/ß)8 barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.


Assuntos
Arabidopsis , alfa-Galactosidase , alfa-Galactosidase/genética , alfa-Galactosidase/química , Rafinose/química , Hidrolases , Filogenia , Galactose
14.
Sci Rep ; 13(1): 5843, 2023 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-37037875

RESUMO

Similar to x-ray imaging, THz imaging will require high power and high resolution to advance relevant applications. Previously demonstrated THz imaging usually experiences one or several difficulties in insufficient source power, poor spectral tunability, or limited resolution from a low-wavelength source. A short-wavelength radiation source in the 5-10 THz is relatively scarce. Although a shorter wavelength improves imaging resolution, widely used imaging sensors, such as microbolometers, Schottky diodes, and photoconductive antennas, are usually not sensitive to detect radiation with frequencies above 5 THz. The radiation power of a high-frequency source becomes a key factor to realize low-noise and high-resolution imaging by using an ordinary pyroelectric detector. Here, we report a successful development of a fully coherent, tunable, > 100-kW-peak-power parametric source at 5.7 THz. It is then used together with a low-cost pyroelectric detector for demonstrating high-resolution 5.7-THz imaging in comparison with 2-THz imaging. To take advantage of the wavelength tunability of the source, we also report spectrally resolved imaging between 5.55 and 5.87 THz to reveal the spectroscopic characteristics and spatial distribution of a test drug, Aprovel.

15.
Nat Commun ; 14(1): 545, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36726015

RESUMO

Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of T = 4 and T = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3-2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central ß-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1', α1'-α1 loop, ß-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral T = 4 and T = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.


Assuntos
Proteínas do Capsídeo , Vírus de RNA , Abelhas , Animais , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Microscopia Crioeletrônica , Conformação Molecular , Montagem de Vírus
16.
J Bacteriol ; 194(22): 6206-16, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22984263

RESUMO

Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5'-phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (BCAA), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The BCAT from Deinococcus radiodurans (DrBCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with L-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT monomer, comprising 358 amino acids, contains large and small domains connected with an interdomain loop. The cofactor PLP is located at the bottom of the active site pocket between two domains and near the dimer interface. The substrate (L-glutamate or KIC) is bound with key residues through interactions of the hydrogen bond and the salt bridge near PLP inside the active site pocket. Mutations of some interaction residues, such as Tyr71, Arg145, and Lys202, result in loss of the specific activity of the enzymes. In the interdomain loop, a dynamic loop (Gly173 to Gly179) clearly exhibits open and close conformations in structures of DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60 °C, than either BCAT from Escherichia coli (eBCAT) or from Thermus thermophilus (HB8BCAT). The dimeric molecular packing and the distribution of cysteine residues at the active site and the molecular surface might explain the resistance to radiation but small thermal stability of DrBCAT.


Assuntos
Deinococcus/enzimologia , Ácido Glutâmico/química , Cetoácidos/química , Transaminases/química , Transaminases/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Espectrofotometria , Especificidade por Substrato
17.
Plant Mol Biol ; 80(4-5): 389-403, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22903596

RESUMO

Cytosolic Oryza sativa glyceraldehyde-3-phosphate dehydrogenase (OsGAPDH), the enzyme involved in the ubiquitous glycolysis, catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate (BPG) using nicotinamide adenine dinucleotide (NAD) as an electron acceptor. We report crystal structures of OsGAPDH in three conditions of NAD-free, NAD-bound and sulfate-soaked forms to discuss the molecular determinants for coenzyme specificity. The structure of OsGAPDH showed a homotetramer form with each monomer comprising three domains-NAD-binding, catalytic and S-loop domains. NAD binds to each OsGAPDH subunits with some residues forming positively charged grooves that attract sulfate anions, as a simulation of phosphate groups in the product BPG. Phe37 not only forms a bottleneck to improve NAD-binding but also combines with Pro193 and Asp35 as key conserved residues for NAD-specificity in OsGAPDH. The binding of NAD alters the side-chain conformation of Phe37 with a 90° rotation related to the adenine moiety of NAD, concomitant with clamping the active site about 0.6 Å from the "open" to "closed" form, producing an increased affinity specific for NAD. Phe37 exists only in higher organisms, whereas it is replaced by other residues (Thr or Leu) with smaller side chains in lower organisms, which makes a greater distance between Leu34 and NAD of E. coli GAPDH than that between Phe37 and NAD of OsGAPDH. We demonstrated that Phe37 plays a crucial role in stabilizing NAD binding or intermediating of apo-holo transition, resulting in a greater NAD-dependent catalytic efficiency using site-directed mutagenesis. Phe37 might be introduced by evolution generating a catalytic advantage in cytosolic GAPDH.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NAD/metabolismo , Oryza/enzimologia , Fenilalanina/metabolismo , Sulfatos/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos
18.
J Biol Chem ; 285(41): 31603-15, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20685646

RESUMO

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)(n), into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67-Thr-69 and Ile-106-Val-112) to interact with (NAG)(n), generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Šalong the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)(n) reveal (NAG)(2), (NAG)(2), and (NAG)(4) in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)(4); in an intermediate state, E145Q/Y227F with a boat-form NAG at the -1 subsite, -1-(NAG); after hydrolysis, E145Q with a chair form -1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between -1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of -1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of -1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)(n) provide new insights into its substrate binding and the mechanistic action.


Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Quitinases/química , Substituição de Aminoácidos , Bacillus cereus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Quitinases/genética , Quitinases/metabolismo , Cristalografia por Raios X , Dissacarídeos/química , Dissacarídeos/genética , Dissacarídeos/metabolismo , Cinética , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
19.
J Biol Chem ; 285(30): 23251-64, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20466731

RESUMO

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.


Assuntos
Aspergillus/enzimologia , Biocatálise , Domínio Catalítico , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-Atividade
20.
Opt Express ; 19(24): 24577-82, 2011 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-22109486

RESUMO

We report forward and backward THz-wave difference frequency generations at 197 and 469 µm from a PPLN rectangular crystal rod with an aperture of 0.5 (height in z) × 0.6 (width in y) mm(2) and a length of 25 mm in x. The crystal rod appears as a waveguide for the THz waves but as a bulk material for the optical mixing waves near 1.54 µm. We measured enhancement factors of 1.6 and 1.8 for the forward and backward THz-wave output powers, respectively, from the rectangular waveguide in comparison with those from a PPLN slab waveguide of the same length, thickness, and domain period under the same pump and signal intensity of 100 MW/cm(2).


Assuntos
Refratometria/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Dinâmica não Linear , Radiação Terahertz
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