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1.
《Biophysical journal》2019,116(10):1941-1951
Light-driven sodium pumps (NaRs) are microbial rhodopsins that utilize light energy to actively transport sodium ions out of the cell. Here, we used targeted mutagenesis and electrophysiological methods in living cells to demonstrate that NaRs can be converted into light-activated cation channels by molecular engineering. Specifically, introduction of the R109Q mutation into the sodium ion pump of Dokdonia eikasta (KR2) results in passive ion conductance, with a high preference for potassium over sodium ions. However, in this mutant, residual active outward pumping of sodium ions competes with passive inward transport of potassium. Channel-like behavior could also be achieved by introduction of other mutations into the KR2 counterion complex, and further, these modifications were transferrable to other NaRs. Combining the R109Q replacement with modifications at position S70 removed the residual sodium pumping and greatly enhanced the channel-like activity. However, passive photocurrents were only observed in leak mutants if the KR2 counterions, D116 and D251, were deprotonated, which was only observed under alkaline conditions. Overall, our results reveal that interactions between R109 and the nearby residues, L75, S70, D116, and D251, prevent passive backflow during ion transport in NaRs. 相似文献
2.
《BBA》2023,1864(3):148980
The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to β-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to β-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin. 相似文献
3.
Deri Morgan Boris Musset Kethika Kulleperuma Susan M.E. Smith Sindhu Rajan Vladimir V. Cherny Régis Pomès Thomas E. DeCoursey 《The Journal of general physiology》2013,142(6):625-640
Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHV1), because the proton concentration is >106 times lower than that of other cations. Here we use “selectivity filter scanning” to elucidate the molecular requirements for proton-specific conduction in hHV1. Asp112, in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp112 by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for “second site suppressor” activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn2+. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction. 相似文献
4.
Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the “Ion-selectivity filter” for sodium ions, while retinal Schiff base acts as the light “Switch and Gate” for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77?K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123. 相似文献
5.
Kyungbin Lee Eun Ji Kim Jaekyum Kim Keun Hee Kim Young Jun Lee Michael J. Lee Kun Ryu Sangyong Shin Jaeyoung Choi Seung Ho Kwon Hyunjoo Lee Jung Kyu Kim Byung-Hyun Kim Bumjoon J. Kim Seung Woo Lee 《Liver Transplantation》2024,14(13):2303803
Traditional challenges of poor cycling stability and low Coulombic efficiency in Zinc (Zn) metal anodes have limited their practical application. To overcome these issues, this work introduces a single metal-atom design featuring atomically dispersed single copper (Cu) atoms on 3D nitrogen (N) and oxygen (O) co-doped porous carbon (CuNOC) as a highly reversible Zn host. The CuNOC structure provides highly active sites for initial Zn nucleation and further promotes uniform Zn deposition. The 3D porous architecture further mitigates the volume changes during the cycle with homogeneous Zn2+ flux. Consequently, CuNOC demonstrates exceptional reversibility in Zn plating/stripping processes over 1000 cycles at 2 and 5 mA cm−2 with a fixed capacity of 1 mAh cm−2, while achieving stable operation and low voltage hysteresis over 700 h at 5 mA cm−2 and 5 mAh cm−2. Furthermore, density functional theory calculations show that co-doping N and O on porous carbon with atomically dispersed single Cu atoms creates an efficient zincophilic site for stable Zn nucleation. A full cell with the CuNOC host anode and high loading V2O5 cathode exhibits outstanding rate-capability up to 5 A g−1 and a stable cycle life over 400 cycles at 0.5 A g−1. 相似文献
6.
The acaricidal properties of 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one isolated from Artemisia iwayomogi and its structural analogues were evaluated against Dermatophagoides farinae and D. pteronyssinus, and their effects were compared with that of the commercial acaricide benzyl benzoate. Based on the 50 % lethal dose (LD50) values against D. farinae, 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (0.82 μg/cm2) was 9.71 times more effective than benzyl benzoate (7.96 μg/cm2), followed by (1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (1.03 μg/cm2), (1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (1.58 μg/cm2), and (1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one oxime (3.05 μg/cm2) in a filter paper bioassay. The acaricidal activities of 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one and its structural analogues against D. pteronyssinus were similar to those against D. farinae. These results demonstrate that naturally occurring A. iwayomogi-isolated 1,7,7-trimethylbicyclo[2.2.1]heptan-2-one and its structural analogues are suitable for the production of natural acaricides against house dust mites. 相似文献
7.
Zeyu He Tao Han Wenfeng Liu Chucheng Zhou Jingyu Sun Ji Zhou Ya-yun Li 《Liver Transplantation》2024,14(11):2303296
Sodium-ion batteries (SIB), as one of the most appealing grid-scale energy storage devices, have to deal with the trade-off between the capacity output and rate performance. Utilizing 3D-printed (3DP) anode materials with hybrid sodium storage mechanism and elevated mass loading is promising yet poorly explored. Herein, the design of a prototype ternary composite is reported, MoS2@Bi/N-doped carbon, as a sodium storage candidate to achieve high reversible capacity (604 mAh g−1 at 0.1 A g−1 with an initial output of 709 mAh g−1) and outstanding rate capability (169.6 mAh g−1 at 15 A g−1), outperforming the state-of-the-art reports. This is realized by delicate structural and interfacial engineering of the composite anode, markedly synergizing the conversion-typed MoS2, alloy-typed Bi, and adsorption-typed N-doped carbon. Theoretical simulations and operando instrumental analysis elaborate the reasons of the boosted electrochemical performance. Encouragingly, a fully 3DP SIB affording an areal mass loading of up to 11.7 mg cm−2 is demonstrated, retaining a capacity of 114 mAh g−1 at 1.0 A g−1. This work would facilitate the design of 3DP SIB devices with the employment of advanced electrodes harnessing hybrid ion storage features. 相似文献
8.
Potential sources of fixed nitrogen in a Colorado desert environment were examined by the acetylene reduction method at the Deep Canyon Desert Research Center, near Palm Desert, California. In field and greenhouse studies all members of the genera Astragalus, Dalea, Lotus, Lupinus, Melilotus, and Prosopis examined formed active nodules (acetylene reduction) with indigenous soil bacteria. No evidence of nodulation was found for Acacia greggii, Cercidium floridum, or Hoffmannseggia microphylla. Lotus tomentellus was estimated to fix 0.1 kg N ha−1 by the time of flowering under field conditions. Several members of the genus Dalea showed substantial rates of acetylene reduction in the greenhouse: D. emoryi, 16.1 + 3.5, D. mollissima, 11.4 + 3.7, D. schottii 2.9 + 1.7, D. spinosa 2.5 + 0.4 μmoles ethylene plant−1 hr−1. In greenhouse assays where water was supplied continuously, blue-green algal-lichen crusts reduced acetylene at an average rate of 11.0 + 5.7 nmoles ethylene cm−2 hr−1 with a maximum of 57.1. But when in situ assays were done following irrigation of a field plot with 2.3 cm of water, much lower activities were observed with a maximum activity of only 6.4 nmoles cm−2 hr−1. 相似文献
9.
《Carbohydrate research》1987,163(1):9-14
Laser-Raman spectra of Na+ kappa-carrageenan, Na+ neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm−1 are reported for solutions in H2O and D2O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm−1, close to the maximum of the symmetrical COS stretching (∼850 cm−1). The symmetrical SO stretch is proposed to occur near 1040 cm−1 and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm−1. The intense band in the region 730–740 cm−1 is ascribed to a complex ring vibration. 相似文献
10.
Hai Li Oleg?A. Sineshchekov Giordano?F.Z. da?Silva John?L. Spudich 《Biophysical journal》2015,109(7):1446-1453
A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H+/Na+ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na+ and H+ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na+ transport and electrogenic H+ transport, respectively, in the presence and absence of Na+. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na+ transport by IAR requires Na+ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. 相似文献
11.
Angela J. Gruber Aysen L. Erdem Grzegorz Sabat Kiyonobu Karata Malgorzata M. Jaszczur Dan D. Vo Tayla M. Olsen Roger Woodgate Myron F. Goodman Michael M. Cox 《PLoS genetics》2015,11(3)
DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. 相似文献
12.
《International journal of biological macromolecules》1987,9(1):54-55
I.r. spectroscopy (the KBr pressed disc method) has been successfully used for the determination of degree of benzylation in chitin. The band at 1550 cm−1 was assigned an internal standard and the 700 cm−1 band was assigned an analytic band. Degree of substitution look up the relative curve of ratio of A700/A1550 against the degree of substitution. 相似文献
13.
You Peng Lijie Zhu Chenyu Li Jingyi Hu Yue Lu Jiatian Fu Fangfang Cui Xiangzhuo Wang Anyuan Cao Qingqing Ji Yahuan Huan Yanfeng Zhang 《Liver Transplantation》2024,14(3):2302510
2D metallic transition-metal dichalcogenides (MTMDCs) have attracted widespread research interest in the exploration of fundamental physical issues and energy-related fields. Although relatively high catalytic activity has been predicted theoretically in the new type MTMDCs-based electrocatalysts, their hydrogen evolution reaction (HER) performance is severely hampered by the insufficient catalytic stability due to structural degradation during long-time use and limited active sites in planar electrode structures. Herein, the scalable synthesis of vertically-oriented 2H-NbS2 nanosheets is reported on low-cost carbon nanotube (CNT) film substrates by a facile chemical vapor deposition route. The 3D vertically-oriented 2H-NbS2 nanosheets present abundant edge active sites and strong interface coupling with CNT thus possessing exceptional mechanical stability. These features impart the 3D nanosheets catalysts with remarkably low overpotentials of ≈55 mV at 10 mA cm−2 and ultra-high exchange current density of ≈1445 µA cm−2, and negligible performance degradation after 200 h operation at the large current density, which are superior to those of other TMDCs-based catalysts. This work hereby provides novel perspectives for the batch synthesis and application of 3D MTMDCs-based electrocatalysts with greatly improved electrocatalytic performance and stability that are needed for practical applications. 相似文献
14.
《Inorganica chimica acta》1986,123(4):231-236
Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm−1) and 575 nm (17 400 cm−1). Anion binding produces characteristic changes in the CD spectra. N3−/SCN− → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm−1) and 365 nm (27 400 cm−1), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm−1) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole. 相似文献
15.
《FEBS letters》1987,220(1):67-73
A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b-559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b-559 in an approximate ratio of 4:2:1 and also contains about one molecule of β-carotene. It binds no plastoquinone-9 or manganese but does contain at least one non-haem iron. In addition to a light-induced signal due to Pheo− seen under reducing conditions, a light-induced P680+ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680+ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680+Pheo or P680Pheo− states are established indicating that the fluorescence emanates from the back reaction between P680+ and Pheo−. 相似文献
16.
Siguang Guo Zhefei Sun Yu Liu Xinbo Guo Haoqin Feng Shi Luo Changhao Wei Yang Zheng Xuming Zhang Kangwoon Kim Haodong Liu Paul K Chu Biao Gao Qiaobao Zhang Kaifu Huo 《Liver Transplantation》2024,14(13):2303876
The manipulation of stress in high-capacity microscale alloying anode materials, which undergo significant volumetric variation during cycling, is crucial prerequisite for improved their cycling capability. In this work, an innovative structural design strategy is proposed for scalable fabrication of a unique 3D highly porous micro structured germanium (Ge) featuring micro-nano hierarchical architecture as viable anode for high-performance lithium-ion batteries (LIBs). The resultant micro-sized Ge, consisting of interconnected nanoligaments and bicontinuous nanopores, is endowed with high activity, decreased Li+ diffusion distance and alleviated volume variation, appealing as an ideal platform for in-depth understanding the relationship between structural design and stress evolution. Such a micro-sized Ge being highly porous delivers a record high initial Coulombic efficiency of 92.5%, large volumetric capacity of 2,421 mAh cm−3 at 1.2 mA cm−2, exceptional rate capability (805.6 mAh g−1 at 10 Ag−1) and cycling stability (over 90% capacity retention after 1000 cycles even at 5 A g−1), largely outperforming the reported Ge-based anodes for LIBs. Furthermore, its underlying Li storage mechanism and stress dispersion behavior are explicitly revealed by combined substantial in situ/ex situ experimental characterizations and theoretical computation. This work provides novel insights into the rational design of high-performance and durable alloying anodes toward high-energy LIBs. 相似文献
17.
《Biophysical journal》2020,118(11):2853-2865
We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl− binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl−-releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl− uptake was faster than that of MF NpHR. These differences in the Cl−-releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl− release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl− uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl−-binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR′ state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl− pumping. 相似文献
18.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,683(1):3-13
Atomic force microscopy (AFM) has been used to probe the surface of a capillary after coating with “soft” polymers, notably polyacrylamides. The aim was the investigation of the efficiency of coverage of the silica surface, so as to reduce or eliminate the electroosmotic flow (EOF), particularly noxious in the separation of macromolecules. The quality of such coating is strongly dependent on two variables: temperature and pH. In the first case, progressively higher temperatures produce open silica patches, where no polymer seems to be bound. The transition from coated to largely uncoated surfaces occurs at 50°C. Also the pH of the polymerizing solution strongly affects the coating efficiency. Since in all coating procedures the monomer solution is not buffered, addition of accelerator (TEMED, N,N,N′N′-tetramethylethylendiamine) induces polymer growth at pH 10–11. These pH values generate hydrolysis of the siloxane bridge anchoring the bifunctional agent (Bind Silane, onto which the polymer chain should grow) to the wall. Thus, coating and de-coating occur simultaneously. Low temperatures during polymer growth (typically 10°C) and buffered solutions (pH 7, titrated after TEMED addition) ensure a most efficient and thorough coating, with virtual elimination of EOF: well coated capillaries exhibit residual EOF values, at pH 10, of the order of 10−7 cm2 V−1 s−1 vs. a standard value for uncoated capillaries of the order of 10−4 cm2 V−1 s−1. The AFM data have been fully confirmed by direct measurement of EOF in coated and uncoated capillaries under an electric field. 相似文献
19.
In the N-terminal domain of thermolysin, two anti-parallel β-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a β-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this β-hairpin structure. 相似文献
20.
Edaphic factors affect the quality of onions (Allium cepa). Two experiments were carried out in the field and glasshouse to investigate the effects of N (field: 0,120 kg ha−1; glasshouse: 0,108 kg ha−1), S (field: 0, 20 kg ha−1; glasshouse: 0, 4.35 kg ha−1) and soil type (clay, sandy loam) on onion quality. A conducting polymer sensor electronic nose (E-nose) was used to classify onion headspace volatiles. Relative changes in the E-nose sensor resistance ratio (%dR/R) were reduced following N and S fertilisation. A 2D Principal Component Analysis (PCA) of the E-nose data sets accounted for c. 100% of the variations in onion headspace volatiles in both experiments. For the field experiment, E-nose data set clusters for headspace volatiles for no N-added onions overlapped (D2= 1.0) irrespective of S treatment. Headspace volatiles of N-fertilised onions for the glasshouse sandy loam also overlapped (D2=1.1) irrespective of S treatment as compared with distinct separations among clusters for the clay soil. N fertilisation significantly (P < 0.01) reduced onion bulb pyruvic acid concentration (flavour) in both experiments. S fertilisation increased pyruvic acid concentration significantly (P < 0.01) in the glasshouse experiment, especially for the clay soil, but had no effect on pyruvic acid concentration in the field. N and S fertilisation significantly (P < 0.01) increased lachrymatory potency (pungency), but reduced total soluble solids (TSS) content in the field experiment. In the glasshouse experiment, N and S had no effect on TSS. TSS content was increased on the clay by 1.2-fold as compared with the sandy loam. Onion tissue N: water-soluble SO42− ratios of between five and eight were associated with greater %dR/R and pyruvic acid concentration values. N did not affect inner bulb tissue microbial load. In contrast, S fertilisation reduced inner bulb tissue microbial load by 80% in the field experiment and between 27% (sandy loam) and 92% (clay) in the glasshouse experiment. Overall, onion bulb quality discriminated by the E-nose responded to N, S and soil type treatments, and reflected their interactions. However, the conventional analytical and sensory measures of onion quality did not correlate with %dR/R. 相似文献