Characterization of the primary photochemistry of proteorhodopsin with femtosecond spectroscopy

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp⁹⁷, is deprotonated) and at pH 6.5 (with Asp⁹⁷ protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of ∼0.2 ps, ∼2 ps, and ∼20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerizaton pathways that lead to the K-state: one with an effective rate of ∼(2 ps)⁻¹ and the other with a rate of ∼(20 ps)⁻¹. At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of ∼0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp²²⁷, which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.     

干旱低磷胁迫对不同品种小麦根系导水率的影响

控制磷素水平,采用控制灌水量(正常供水、中度及重度干旱胁迫)的盆栽试验法,选择抗旱性小麦品种陕合6号(W1)和水分敏感型品种郑引1号(W2)为供试材料。用压力室法测定了三叶期的两品种小麦根系导水率(LPr)的变化规律。结果表明：陕合6号,在有磷正常供水处理( PH)下具有较高的导水率,干旱胁迫时LPr降低较少,且复水后有较强的恢复能力。郑引1号, PH的LPr值相对较小,干旱导致的根系导水率下降非常突出,复水后的恢复能力也较弱。另外,干旱胁迫对小麦苗期根系导水率的影响大于磷胁迫对其导水率的影响,且两品种小麦无磷止常供水处理(-PH)的LPr分别为 PH的31．9%和53．6％,即磷对前者LPr的影响大于后者。     

Enhanced photoluminescence of the Ca0.8Zn0.2TiO3:0.05% Pr3+ phosphor by optimized hydrothermal conditions

The red‐emitting phosphor Ca_0.8Zn_0.2TiO₃:Pr³⁺ was synthesized using an ethylene glycol (EG)‐assisted hydrothermal method. The effects of additional amounts of and order of adding EG, plus hydrothermal temperature, time, and pH on the composition, morphology and optical properties of the titanate phosphors were studied. The crystalline phases of the titanate phosphors were confirmed to be constituted of a series of co‐existing CaTiO₃, Zn₂TiO₄ and Ca₂Zn₄Ti₁₆O₃₈ compounds in various proportions that were visualized using an X‐ray diffractometer (XRD). The optical properties of the phosphors were studied using photoluminescence spectra and UV–visible spectroscopy. The results show that the impurities Zn₂TiO₄:Pr³⁺ and Ca₂Zn₄Ti₁₆O₃₈:Pr³⁺ significantly contributed to the enhancement of an absorption band around 380 nm. The optimum Ca_0.8Zn_0.2TiO₃:Pr³⁺ phosphor consisting of appropriate amounts of CaTiO₃, Ca₂Zn₄Ti₁₆O₃₈ and Zn₂TiO₄ in three phases was achieved by controlling the hydrothermal conditions, and the obtained red phosphor exhibited the highest red emission (¹D₂ → ³H₄ transition of Pr³⁺) with an ideal chromaticity coordinate located at (x = 0.667, y = 0.332) under 380 nm excitation.     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

玫烟色棒束孢Pr1酶活力、Pr1酶基因表达量与其毒力的相关性

丝氨酸弹性凝乳蛋白酶Pr1是一类能高效降解昆虫体壁蛋白的重要酶,其活力与虫生真菌的毒力有很大的关系。探索玫烟色棒束孢不同菌株Pr1酶活力、Pr1蛋白酶基因表达量与毒力的相关性对该菌的应用具有重要的意义。文中采用专一性短肽底物Suc-Ala-Ala-Pro-Phe-pNA和荧光定量PCR分别测定了玫烟色棒束孢不同菌株的Pr1酶活和Pr1基因的表达量,并采用坡塔喷雾法测定了供试菌株对桃蚜的毒力。结果表明:不同供试菌株Pr1蛋白酶活力与其毒力的线性回归方程为y=3.64x+0.62,R~2=0.432,两者呈正相关;供试菌株Pr1酶活力、Pr1基因表达量与毒力的回归方程为y=0.236+10.833x_1–0.039x_2 (x_1=Pr1酶活力,x_2=Pr1基因表达量),R~2=0.568,说明线性拟合方程能很好地反映原始数据;序列相关系数D-W为2.444,在0.05水平上相关显著,表明Pr1酶活力、Pr1基因表达量对毒力有显著影响;VIF=12.705表明Pr1酶活力、Pr1基因表达量存在中度多重共线性。因此建议将Pr1蛋白酶的酶活力和Pr1基因表达量作为菌株毒力筛选时的重要指标。     

The impact of the phytochromes on photosynthetic processes

This review describes the phytochrome system in higher plants and cyanobacteria and its role in regulation of photosynthetic processes and stress protection of the photosynthetic apparatus. A relationship between the content of the different phytochromes, the changes in the ratios of the physiologically active forms of phytochromes to their total pool and the resulting influence on photosynthetic processes is reviewed. The role of the phytochromes in the regulation of the expression of genes encoding key photosynthetic proteins, antioxidant enzymes and other components involved in stress signaling is elucidated.     

Binding kinetics,potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir

Background

GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.

Methods

Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.

Results

GS-9256 and vedroprevir had measured K_i values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; K_i values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 10⁵ M^− 1 s^− 1 and 1 × 10⁶ M^− 1 s^− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10^− 5 s^− 1 and 2.6 × 10^− 4 s^− 1 were determined.

Conclusions

GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.

General Significance

The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330