Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.     

Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein

We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K_d) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.     

Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.     

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.     

Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Conformational changes of a viral capsid protein. Thermodynamic rationale for proteolytic regulation of bacteriophage T4 capsid expansion, co-operativity, and super-stabilization by soc binding.

We have used differential scanning calorimetry in conjunction with cryo-electron microscopy to investigate the conformational transitions undergone by the maturing capsid of phage T4. Its precursor shell is composed primarily of gp23 (521 residues): cleavage of gp23 to gp23* (residues 66 to 521) facilitates a concerted conformational change in which the particle expands substantially, and is greatly stabilized. We have now characterized the intermediate states of capsid maturation; namely, the cleaved/unexpanded, state, which denatures at tm = 60 degrees C, and the uncleaved/expanded state, for which tm = 70 degrees C. When compared with the precursor uncleaved/unexpanded state (tm = 65 degrees C), and the mature cleaved/expanded state (tm = 83 degrees C, if complete cleavage precedes expansion), it follows that expansion of the cleaved precursor (delta tm approximately +23 degrees C) is the major stabilizing event in capsid maturation. These observations also suggest an advantage conferred by capsid protein cleavage (some other phage capsids expand without cleavage): if the gp23-delta domains (residues 1 to 65) are not removed by proteolysis, they impede formation of the stablest possible bonding arrangement when expansion occurs, most likely by becoming trapped at the interface between neighboring subunits or capsomers. Icosahedral capsids denature at essentially the same temperatures as tubular polymorphic variants (polyheads) for the same state of the surface lattice. However, the thermal transitions of capsids are considerably sharper, i.e. more co-operative, than those of polyheads, which we attribute to capsids being closed, not open-ended. In both cases, binding of the accessory protein soc around the threefold sites on the outer surface of the expanded surface lattice results in a substantial further stabilization (delta tm = +5 degrees C). The interfaces between capsomers appear to be relatively weak points that are reinforced by clamp-like binding of soc. These results imply that the "triplex" proteins of other viruses (their structural counterparts of soc) are likely also to be involved in capsid stabilization. Cryo-electron microscopy was used to make conclusive interpretations of endotherms in terms of denaturation events. These data also revealed that the cleaved/unexpanded capsid has an angular polyhedral morphology and has a pronounced relief on its outer surface. Moreover, it is 14% smaller in linear dimensions than the cleaved/expanded capsid, and its shell is commensurately thicker.     

Effects of ultraviolet radiation on protein content,respiratory electron transport system (ETS) activity and superoxide dismutase (SOD) activity of Antarctic plankton

Depletion of stratospheric ozone causes a significant increase in UV radiation in the Antarctic regions. Its effects include DNA damage, as well as impairment of photosynthesis, respiration, protein synthesis and other metabolic functions. Defence systems of cells are directed against free oxygen radicals liberated through UV radiation. One of their main components of defence systems are superoxide dismutases (SODs). The effects of ultraviolet radiation A and B (UVAR and UVBR) on protein synthesis, respiratory electron transfer (ETS) activity and superoxide dismutase (SOD) activity in Antarctic plankton were examined. Samples were taken in the Gerlache Strait (Antarctica). Three stations were situated in an area, which showed a Cryptomonas bloom. Two stations were located in areas having a bloom of green nanoflagellates. Samples were exposed for 3 h to photosynthetically active radiation (PAR), or to PAR + UVAR or to PAR + UVAR + UVBR, under fixed experimental irradiances. UVBR inhibited protein synthesis and ETS activity, and enhanced SOD activity. UVAR enhanced protein synthesis and ETS activity, and inhibited SOD activity. Samples, which had received more solar radiation prior to experiments showed less inhibition of protein synthesis by experimental UVBR, which suggests acclimation to ambient radiation. Cryptomonas-dominated stations showed less SOD activity than the green flagellate-dominated stations, which might be related to the protection conferred by their phycoerythrin.     

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.     

增强UV-B辐射对大豆胚轴DNA损伤、修复和蛋白质含量的影响

大气平流层臭氧层减薄引起到达地表的 UV- B辐射增强。为探讨在增强 UV- B辐射下植物细胞 DNA的损伤修复和蛋白质含量的关系 ,利用 3H- Td R掺入法 ,研究了在 8.2 2 k J/(m2 d)和 12 .4 2 k J/(m2 d) U V- B辐射 (相当于兰州地区大气平流层臭氧减薄约 12 %和 2 0 % )胁迫下 ,大豆胚轴细胞 DNA合成和非按期合成 (UDS)变化 ,并测定了胚轴蛋白质含量变化 ,结果显示 ,UV- B辐射导致 DNA损伤 ,并诱导了 DNA损伤的修复 ,胚轴细胞 UDS效应增强 ,U DS指数增大。低 UV- B辐射强度下 ,胚轴蛋白质含量增加 ,可能是 U V- B诱导了一些与抗性有关的基因表达 ,导致一些新的与抗性有关的蛋白质合成 ;在高强度 UV- B辐射下 ,U DS指数与低强度辐射下无显著差异 (P=0 .0 5 ) ,但蛋白含量较低强度辐射下显著下降 (P=0 .0 5 ) ,说明高强度 UV- B辐射加重了 DNA损伤 ,而修复并未加强 ,并且高强度辐射抑制基因的正常表达和蛋白质合成。这些蛋白质的合成可能与大豆对 UV- B辐射的抗性有关。     

HBXIP,a binding protein of HBx,regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity

To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.     

Nonuniform Ionic and Electronic Transport of Ceramic and Polymer/Ceramic Hybrid Electrolyte by Nanometer‐Scale Operando Imaging for Solid‐State Battery

Replacing the liquid electrolyte in lithium batteries with solid‐state ion conductor is promising for next‐generation energy storage that is safe and has high energy density. Here, nanometer‐resolution ionic and electronic transport imaging of Li₃PS₄ (LPS), a solid‐state electrolyte (SSE), is reported. This nm resolution is achieved by using a logarithm‐scale current amplifier that enhances the current sensitivity to the fA range. Large fluctuations of ion current—one to two orders of magnitude on the LPS and on the LPS region of a polymer/LPS bulk hybrid SSE—that must be mitigated to eliminate Li dendrite formation and growth, are found. This ion current fluctuation is understood in terms of highly anisotropic transport kinetic barriers along the different crystalline axes due to different grain orientations in the polycrystalline and glass ceramic materials. The results on the bulk hybrid SSE show a sharp transition of ionic and electronic transport at the LPS/polymer boundary and decreases in average ionic current with decreasing polyimine particle size and with extensive cycling. The results elucidate the mechanism of polyimine extension into interparticles to prevent Li dendrite growth. This work opens up novel characterization of charge transport, which relates to Li plating and stripping for solid‐state‐batteries.     

Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH,temperature, and ligand binding

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in different forms of human hemoglobin (Hb) at pH 5–10 and temperatures of 10–63°C. The pH dependence of the H-D exchange rate fits the EX2 mechanism. At 10–30°C, there are two pH-dependent conformers of liganded Hb forms, the fluctuation probability being lower for the alkaline conformer. The differences between the conformers disappear at 40°C, where a third conformer, with a higher probability of local fluctuations, appears. Deoxyhemoglobin has no pH-dependent conformers in the pH range 6–9 at 20°C, and the probability of local fluctuations is considerably decreased compared to the acid conformer of liganded Hb. The destabilization of the liganded Hb structure by decreasing the pH to 5.0 at 20°C or increasing the temperature to 50–60°C at pH 7.1 enhances global fluctuations of the native structure ensuring the H-D exchange of slowly exchanging NH atoms. The mechanisms of local and high-temperature global fluctuations, as well as the possible similarity between the two pH-dependent conformers of liganded Hb and its functional R and R2 states revealed by X-ray analysis and NMR spectroscopy, are discussed.     

Influence of UV-B radiation on developmental changes, ethylene, CO2 flux and polyamines in cv. Doyenne d'Hiver pear shoots grown in vitro

In vitro shoots of cv. Doyenne ďHiver pear ( Pyrus communis L.) were irradiated under controlled environments for 6 h per day at 5 different levels of biologically effective UV-B radiation (UV-B_BE). UV-B exposure caused a progressive increase in apical necrosis above background levels and stimulated leaf abscission. Shoots grown for 2 weeks at 7. 8 mol m⁻² day ⁻¹ of photosynthetic photon flux (PPF) and treated with 8. 4 or 12. 0 kJ m⁻² day ⁻¹ UV-B_BE produced up to 4 times more ethylene than those given 2. 2 or 5. 1 kJ m⁻² day⁻¹ UV-B_BE or untreated controls. Exposure of shoots to 12 kJ m⁻² day ⁻¹ of UV-B_BE caused an increase in free putreseine content after 4 to 14 days of irradiation. Shoots showed a decrease in CO₂ uptake after 3 days of UV-B: thereafter, they appeared to recover their photosynthetic capacity. Under typical PPF conditions used in micropropagation (90 μmol m⁻² S⁻¹). 8. 4 kJ m⁻² day ⁻¹ of UV-B radiation was injurious to realatively tender tissues of in vitro pear shoots: increasing the level of UV-B_BE to 12 kJ m⁻² day⁻¹ produced even more adverse effects.     

Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed,pre-transition-state conformations

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.