A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.     

Immunogold localization of phytoene desaturase in higher plant chloroplasts

In situ location of phytoene desaturase, a key enzyme in the carotenoid biosynthesis pathway, has been investigated in chloroplasts from higher plants. For this purpose, an antiserum has been raised against the phytoene desaturase from the cyanobacterium Synechococcus PCC 7942 overexpressed in E. coli . The specifity of this antiserum was demonstrated by inhibition of the enzymatic desaturation reaction in vitro. The antiserum was further purified and immunoabsorbed with E. coli proteins. The resulting IgG-fraction was tested by western blotting against membrane proteins from chloroplasts of tobacco ( Nicotiana tabacum L. cv. Samsun) and spinach ( Spinacia oleracea L. cv. Atlanta). Apparent molecular masses of immunoreactive proteins were 62 and 64 kDa. A western blot of different membrane fractions of spinach chloroplasts (inner and outer envelopes, and thylakoids) indicated a localization of the phytoene desaturase in thylakoids. A post embedding immunogold microscopy procedure was employed. In these experiments the main labelling (79%) was associated with thylakoid membranes of tobacco chloroplasts. Of the counted colloidal gold particles, 16% were found in the stroma. Only 5% were detected in the envelope membranes. These results give clear evidence that at least the majority of phytoene desaturase molecules is localized within thylakoid membranes of higher plant chloroplasts and that the presence of the enzyme in the envelope is of minor significance.     

Cd、Pb及其复合污染对烤烟叶片生理生化及细胞亚显微结构的影响

本研究以水培的烤烟给予不同浓度的Cd、Pb及其复合物处理10d后的烟叶为材料,分析了烟叶过氧化氢酶、硝酸还原酶的活性变化,测试了烟叶可溶性糖含量的变化情况,通过透射电子显微镜观察到了Cd和Pb对烟叶叶肉细胞亚显微结构的改变,特别是对叶绿体、线粒体和细胞核结构的损伤情况进行了详细观察。并探讨其毒害机理。研究结果表明：1)烟叶过氧化氢酶的活性剧烈地被Cd抑制;而随着Pb浓度的增加,其活性则表现为先增加后减弱的变化。2)Cd对硝酸还原酶活性的影响表现为先刺激增强,当Cd浓度超过50mg·L-1后,Cd剧烈地抑制其活性,当Cd浓度为200mg·L-1时,其活性几乎为零;Pb抑制烟叶硝酸还原酶的活性,仅在1000mg·L-1时出现一个低于正常活性的抗性峰。3)烟叶可溶性糖含量对Cd、Pb及其复合污染非常敏感,在较低浓度的污染处理时,其含量就明显下降,烟叶可溶性糖含量的变化可作为监测Cd、Pb污染的指标。4)Cd对烟叶叶肉细胞亚显微结构具有较强的损伤诱变作用,对细胞核、叶绿体和线粒体造成不可逆转的伤害,破坏了细胞正常生理活动所需的结构基础。电镜观察表明Cd严重地破坏细胞的膜结构。这可能是由于Cd离子与蛋白质结合而使蛋白质变性,从而使得以蛋白质为重要组成成份之一的膜的结构改变,功能丧失。5)在细胞膜的外面可以看到大量的Pb沉积粒,细胞膜可以阻止部分Pb进入原生质体内部,但在细胞质和叶绿体中仍可看到Pb沉积粒。Pb同样的损伤叶绿体、线粒体、细胞核的亚显微结构。     

Temperature-dependent binding to the thylakoid membranes of nuclear-coded chloroplast heat-shock proteins

Two nuclear-coded heat-shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post-translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25 degrees C the 22-kDa and 25-kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat-shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non-binding to the binding status is comparatively sharp and occurs between 36 degrees C and 40 degrees C in the variety 'Rosa Krone'. The transition temperature has been determined at 38 degrees C for 'Rosa Krone' and at 40 degrees C for the variety 'Golf'. At 42 degrees C, 15-min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4-6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat-shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat-shock proteins.     

Thermal and chemical unfolding and refolding of a eukaryotic sodium channel

Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with “denatured” channels, retaining significant helical secondary structure even at 77 °C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.     

Association of TMV coat protein with chloroplast membranes in virus-infected leaves

Summary The polypeptide composition of extracts of chloroplasts from tobacco leaves systemically infected with different strains of Tobacco Mosaic Virus (TMV) was analyzed by one- and two-dimensional gel electrophoresis. There were no changes in the protein profiles of chloroplasts from infected leaves when compared to control leaves except for the presence of coat protein (CP) of TMV, identified by immunoblotting. When protease-treated intact chloroplasts isolated on Percoll gradients were osmotically disrupted the CP could be detected in both stroma and membrane fractions. The majority of the CP associated with the thylakoid membranes (about 1–5% of the total thylakoid proteins) was in the form of free molecules while stroma contained aggregated or assembled CP (about 0.1% of the soluble proteins). Thylakoid-associated CP was insensitive to protease digestion unless the membranes were first treated with a detergent, indicating that the CP was embedded inside or otherwise complexed with the thylakoid membranes.Chloroplasts isolated from leaves infected with TMV-PV42, a symptomless strain, contained approximately 10–50 times less CP than did chloroplasts isolated from leaves bearing mosaic symptoms induced by other strains of TMV (U1, PV230 or PV39). A possible role of CP in symptom development is discussed.     

Chloroplast grana membrane carboxyl groups: their involvement in membrane association

Chloroplast membrane carboxyl groups were modified by carbodiimide activation followed by glycine methyl ester substitution, leaving the derivatized group uncharged. This charge alteration induced a number of effects similar to addition of salts to control chloroplasts suspended in a low salt medium. These include: (a) restacking or multiple membrane association in low salt-treated chloroplasts that lack grana stacks, (b) protection against polycation inhibition of photosystem I electron transfer, (c) reduction of the amount of polycations bound to the membranes, and (d) increased 90° light scattering due to membrane conformational changes. Carboxyl modification also altered acid-induced conformational changes.     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

Conformational fluctuations in chloroplasts

Characteristic features of conformational changes in chloroplasts have been studied by microfilm technique at BMI-13 installation. The oscillatory character of changes in chloroplast cross-section area in time, determined earlier by the tele-lazer microscopy technique, has been confirmed by our data. When chloroplasts are simultaneously seen in the microscope it is possible to observe their synchronous oscillation. The stationarity check of conformational changes shows that is a non-stationary process in the general case. So it is concluded that the standard technique of analysing accidental non-stationary processes can be used for study of conformational changes in chloroplasts provided that the corresponding parts of their kinetics are selected or trends are eliminated depending on the type of non-stationarity.     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

Calculation of rigid-body conformational changes using restraint-driven Cartesian transformations

We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.     

Investigating the production of foreign membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal oxidase

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5'UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.     

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.     

Blue Native/SDS‐PAGE and iTRAQ‐Based Chloroplasts Proteomics Analysis of Nicotiana tabacum Leaves Infected with M Strain of Cucumber Mosaic Virus Reveals Several Proteins Involved in Chlorosis Symptoms

Virus infection in plants involves necrosis, chlorosis, and mosaic. The M strain of cucumber mosaic virus (M‐CMV) has six distinct symptoms: vein clearing, mosaic, chlorosis, partial green recovery, complete green recovery, and secondary mosaic. Chlorosis indicates the loss of chlorophyll which is highly abundant in plant leaves and plays essential roles in photosynthesis. Blue native/SDS‐PAGE combined with mass spectrum was performed to detect the location of virus, and proteomic analysis of chloroplast isolated from virus‐infected plants was performed to quantify the changes of individual proteins in order to gain a global view of the total chloroplast protein dynamics during the virus infection. Among the 438 proteins quantified, 33 showed a more than twofold change in abundance, of which 22 are involved in the light‐dependent reactions and five in the Calvin cycle. The dynamic change of these proteins indicates that light‐dependent reactions are down‐accumulated, and the Calvin cycle was up‐accumulated during virus infection. In addition to the proteins involved in photosynthesis, tubulin was up‐accumulated in virus‐infected plant, which might contribute to the autophagic process during plant infection. In conclusion, this extensive proteomic investigation on intact chloroplasts of virus‐infected tobacco leaves provided some important novel information on chlorosis mechanisms induced by virus infection.     

Expression and characterization of antimicrobial peptides Retrocyclin‐101 and Protegrin‐1 in chloroplasts to control viral and bacterial infections

Retrocyclin‐101 (RC101) and Protegrin‐1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV‐1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His‐tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%–38% and 17%～26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa–like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6‐fold higher yield of RC101 than purification by affinity chromatography using His‐tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further preclinical studies.     

毒素蛋白Colicin E1与磷脂膜的相互作用

多肽及蛋白质的插膜机制是目前分子生物学、细胞生物学研究中十分活跃的领域之一。本文通过荧光、圆二色等波谱学技术,深入地探讨了处于不同构象状态的毒素蛋白分子与磷脂膜作用后的构象变化。结果表明：带负电荷的磷脂膜对处于不同构象状态的ＣｏｌｉｃｉｎＥ１分子的二级结构有较强的诱导作用;这种作用是电荷依赖性的。处于不同构象状态的毒素蛋白分子在磷脂膜的诱导下均可不同程度恢复其天然状态下插膜时的构象。不同磷脂对ＣｏｌｉｃｉｎＥ１分子诱导的强弱依次为ＤＭＰＧ＞ＤＭＰＥ＞ＤＭＰＣ。ＣｏｌｉｃｉｎＥ１分子与磷脂膜的结合是紧密的,结合后的蛋白质有较强的抗变性能力。     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Effects of Simulated Acid Rain on Chloroplast Ultrastructure of Primary Leaves of Phaseolus Vulgaris

The ultrastructure of chloroplasts in the primary leaf of 10-d-old bean plants (Phaseolus vulgaris L., cv. Cheren Starozagorski) was studied 3, 5, 24, 48, 72 and 168 h after a single treatment with simulated acid rain (pH 2.4, 2.2, 2.0 and 1.8). Different changes in chloroplast structure till full destruction of organelles were traced. A determining factor for these changes was the histological localization of chloroplasts. In the chloroplasts of palisade parenchyma different degrees of expansion of thylakoids (3, 5, and 24 h after the single treatment), and conformational changes of the inner membrane system (48, 72 and 168 h) were observed. The chloroplasts of spongy parenchyma showed a significantly higher degree of structure resistance. The expansion of thylakoids was weak and did not depend on the duration of treatment. The ultrastructural changes of chloroplasts confirmed relative resistance of this species till pH 2.0.     

Comparison of the Molecular Weights of Proteins Synthesized by Isolated Chloroplasts with Those Which Appear during Greening in Zea mays

The proteins of prolamellar bodies of etioplasts and of thylakoid membranes of greening and mature chloroplasts from Zea mays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three classes of proteins were distinguished: those present in etioplasts and disappearing during greening, those absent in etioplasts and appearing during greening, and those present in both etioplasts and chloroplasts. The largest number of proteins belonged to this last class.The molecular weights of chloroplast thylakoid proteins were compared to the molecular weights of the membrane-associated proteins synthesized by isolated, mature chloroplasts. Thirteen of the 15 to 20 membrane-bound proteins made by isolated chloroplasts corresponded in size to proteins present in chloroplasts. Most of the 13 are present in both etioplasts and chloroplasts although a few were the same size as proteins which increase during greening. Production of most of the membrane proteins made in the plastids is not stringently regulated by light in vivo. The polypeptide subunits of the light-harvesting pigment-protein complex, the most abundant proteins of the chloroplast thylakoids, were absent from etioplasts. They were not synthesized by isolated chloroplasts.