Structural Analysis of A-Protein of Cucumber Green Mottle Mosaic Virus and Tobacco Mosaic Virus by Synchrotron Small-Angle X-Ray Scattering

The size and shape of A-protein of tobacco mosaic virus coat protein (TMVP) and cucumber green mottle mosaic virus coat protein (CGMMVP) were evaluated by means of small-angle X-ray scattering (SAXS) using a synchrotron radiation source, complemeted by electron microscopic observations. The results imply that TMV and CGMMV A-proteins are composed of three and two subunits, respectively, stacked in the shape of an isosceles triangular prism at lower ionic strength. Considering the difference of the A-protein structure at higher and lower ionic strength, the globular core structure was proposed as a subunit which might be modeled as a thin isosceles triangular prism composed of four globular cores joined by rather flexible segments. These cores correspond probably to four helical regions in a subunit, and rearrange their relative positions according to the external conditions. A slight rearrangement of core positions in a subunit may result in the formation of A-proteins of various shapes.     

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.     

Molecular Characterization and Distribution of Cucumber green mottle mosaic virus in China

Cucumber green mottle mosaic virus (CGMMV) is an emerging virus on watermelon in China. We report here the almost complete nucleotide sequence and the characterization of the genome of a Chinese isolate (CGMMV-LN). Nucleotide sequence comparisons showed CGMMV-LN is closely related to CGMMV-KOM, with 99.4% identity. On the basis of the nucleotide sequence, a digoxigenin-labelled cDNA probe CG, complementary to the replicase gene region of CGMMV was synthesized. The specificity and sensitivity of the probe was tested. The detection limit of the method was equivalent to 0.8 μg fresh tissues infected by CGMMV. Two hundreds and eighteen watermelon samples collected from different regions in China during 2006–2007 were tested by this method. The distribution pattern of CGMMV in China during these years was revealed. The virus has spread in five provinces of China so far, including Liaoning, Hebei, Guangdong, Hubei and Shandong and might be an increasing tendency, which provides important information for CGMMV control in China.     

Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection

In watermelon, grafting of seedlings to rootstocks is necessary because watermelon roots are less viable than the rootstock. Moreover, commercially important watermelon varieties require disease-resistant rootstocks to reduce total watermelon yield losses due to infection with viruses such as cucumber green mottle mosaic virus (CGMMV). Therefore, we undertook to develop a CGMMV-resistant watermelon rootstock using a cDNA encoding the CGMMV coat protein gene (CGMMV-CP), and successfully transformed a watermelon rootstock named gongdae. The transformation rate was as low as 0.1–0.3%, depending on the transformation method used (ordinary co-culture vs injection, respectively). However, watermelon transformation was reproducibly and reliably achieved using these two methods. Southern blot analysis confirmed that the CGMMV-CP gene was inserted into different locations in the genome either singly or multiple copies. Resistance testing against CGMMV showed that 10 plants among 140 T₁ plants were resistant to CGMMV infection. This is the first report of the development by genetic engineering of watermelons resistant to CGMMV infection.     

High genetic stability in natural populations of the plant RNA virus tobacco mild green mosaic virus

Summary Quantitative studies on the genetic variation of plant viruses are very scarce, in spite of their theoretical and applied importance. We report here on the genetic variability of field isolates of the plant RNA virus tobacco mild green mosaic virus (TMGMV) naturally infecting the wild plantNicotiana glauca Grah. The populations studied were composed of a high number of haplotypes. Two main features are found regarding TMGMV variation: First, there is no correlation between genetic proximity of isolates and geographic proximity of the sites from which they were obtained; and second, the estimated divergence among haplotypes is low, and values are maintained regardless of the scale of the distance between the sites from which the isolates come. No comparable studies have been done with a plant RNA virus, and these two features seem to be unique for this system as compared with other RNA viruses.     

Transgenic <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants resistant to cucumber green mottle mosaic virus based on RNA silencing

RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection. The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing is strong and efficient.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems.     

Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.     

小角X-射线散射法解析多结构域蛋白质或复合物的溶液结构

随着同步辐射装置的建设与发展及各种建模方法的产生与完善,小角X-射线散射（small angle X-ray scattering,SAXS）法已经逐渐成为结构生物学中的一种重要的工具。SAXS可以用于研究溶液中生物大分子的结构及构象变化,蛋白质的组装、折叠等动态过程。本文对SAXS的基本原理、常用的研究技术和建模方法及其应用进行了综述。     

Indices of water deficit in susceptible and resistant cucumbers in response to infection by cucumber mosaic virus

Indices of water deficit were determined under conditions of non-limited water supply in cucumber mosaic virus (CMV)-infected seedlings of the susceptible cv. Bet Alpha. An increase in the concentration of soluble solids, decrease of water and osmotic potentials, and increase of proline concentration were found in the CMV-infected cotyledons. In the cv. Shimshon, which is resistant to CMV, virus infection caused only a slight change in the concentration of the soluble solids and in the osmotic potential; water potential and proline content were not affected. Concomitantly, infectivity of cotyledons by CMV was much lower in the tolerant cv. Shimshon than in the susceptible cv. Bet Alpha. The possible association of water deficit with virus-induced growth retardation is discussed.     

Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Using fluorescence correlation spectroscopy (FCS), we have established an in vitro assay to study RNA dynamics by analyzing fluorophore binding RNA aptamers at the single molecule level. The RNA aptamer SRB2m, a minimized variant of the initially selected aptamer SRB-2, has a high affinity to the disulfonated triphenylmethane dye sulforhodamine B. A mobility shift of sulforhodamine B after binding to SRB2m was measured. In contrast, patent blue V (PBV) is visible only if complexed with SRB2m due to increased molecular brightness and minimal background. With small angle X-ray scattering (SAXS), the three-dimensional structure of the RNA aptamer was characterized at low resolution to analyze the effect of fluorophore binding. The aptamer and sulforhodamine B-aptamer complex was found to be predominantly dimeric in solution. Interaction of PBV with SRB2m led to a dissociation of SRB2m dimers into monomers. Radii of gyration and hydrodynamic radii, gained from dynamic light scattering, FCS, and fluorescence cross-correlation experiments, led to comparable conclusions. Our study demonstrates how RNA-aptamer fluorophore complexes can be simultaneously structurally and photophysically characterized by FCS. Furthermore, fluorophore binding RNA aptamers provide a tool for visualizing single RNA molecules.     

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co²⁺, glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co²⁺ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.     

Conformational Flexibility of Ubiquitin-Modified and SUMO-Modified PCNA Shown by Full-Ensemble Hybrid Methods

Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.     

The equilibrium unfolding intermediate observed at pH 4 and its relationship with the kinetic folding intermediates in green fluorescent protein

Folding mechanisms of a variant of green fluorescent protein (F99S/M153T/V163A) were investigated by a wide variety of spectroscopic techniques. Equilibrium measurements on acid-induced denaturation of the protein monitored by chromophore and tryptophan fluorescence and small-angle X-ray scattering revealed that this protein accumulates at least two equilibrium intermediates, a native-like intermediate and an unfolding intermediate, the latter of which exhibits the characteristics of the molten globule state under moderately denaturing conditions at pH 4. To elucidate the role of the equilibrium unfolding intermediate in folding, a series of kinetic refolding experiments with various combinations of initial and final pH values, including pH 7.5 (the native condition), pH 4.0 (the moderately denaturing condition where the unfolding intermediate is accumulated), and pH 2.0 (the acid-denaturing condition) were carried out by monitoring chromophore and tryptophan fluorescence. Kinetic on-pathway intermediates were accumulated during the folding on the refolding reaction from pH 2.0 to 7.5. However, the signal change corresponding to the conversion from the acid-denatured to the kinetic intermediate states was significantly reduced on the refolding reaction from pH 4.0 to pH 7.5, whereas only the signal change corresponding to the above conversion was observed on the refolding reaction from pH 2.0 to pH 4.0. These results indicate that the equilibrium unfolding intermediate is composed of an ensemble of the folding intermediate species accumulated during the folding reaction, and thus support a hierarchical model of protein folding.     

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.