[Structure of Potato Virus A Coat Protein Particles and Their Dissociation].

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to ß-conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit-detergent complexes consisting of 10-12 small particles joined together like "beads on a string". Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into ß-structures has been suggested.

Structural Properties of Potexvirus Coat Proteins Detected by Optical Methods.

It has been shown by X-ray analysis that cores of coat proteins (CPs) from three potexviruses, flexible helical RNA-containing plant viruses, have similar α-helical structure. However, this similarity cannot explain structural lability of potexvirus virions, which is believed to determine their biological activity. Here, we used circular dichroism (CD) spectroscopy in the far UV region to compare optical properties of CPs from three potexviruses with the same morphology and similar structure. CPs from Alternanthera mosaic virus (AltMV), potato aucuba mosaic virus (PAMV), and potato virus X (PVX) have been studied in a free state and in virions. The CD spectrum of AltMV virions was similar to the previously obtained CD spectrum of papaya mosaic virus (PapMV) virions, but differed significantly from the CD spectrum of PAMV virions. The CD spectrum of PAMV virions resembled in its basic characteristics the CD spectrum of PVX virions characterized by molar ellipticity that is abnormally low for α-helical proteins. Homology modeling of the CP structures in AltMV, PAMV, and PVX virions was based on the known high-resolution structures of CPs from papaya mosaic virus and bamboo mosaic virus and confirmed that the structures of the CP cores in all three viruses were nearly identical. Comparison of amino acid sequences of different potexvirus CPs and prediction of unstructured regions in these proteins revealed a possible correlation between specific features in the virion CD spectra and the presence of disordered N-terminal segments in the CPs.

[Disordered regions in C-domain structure of influenza virus M1 protein].

Influenza virus matrix M1 protein is one of the main structural components of the virion performing also many different functions in infected cell. X-ray analysis data with 2.08 angstrom resolution were obtained only for the N-terminal part of M1 protein molecule (residues 2-158) but not for its C-terminal domain (159-252). In the present work M1 protein of A/Puerto Rico/8/34 (H1N1) virus strain in acidic solution was investigated with the help of tritium bombardment. Tritium label incorporation into M1 protein domains preferentially labeled the C-domain and inter-domain loops. Analytical centrifugation and dynamic light scattering experiments demonstrated increased hydrodynamic parameters (diameter) that may be explained by low degree of M1 structural organization. Computational analysis of M1 protein by intrinsic disorder predictions methods also demonstrated the presence of unfolded regions mostly in the C-domain and inter-domain loops. It is suggested, that influenza virus M1 polyfunctionality in infected cell is determined by its tertiary structure plasticity which in its turn results from the presence of unstructured regions.

[Comparative study of structural stabylity of potato virus X coat protein molecules in solution and in the virus particles].

With help of several optical methods and differential scanning calorimetry we studied the structure and stability of molecules of coat protein (CP) of filamentous of potato virus X (PVX) in free state and in the virions. According to the results of all these methods, at room temperature (25 degrees C) free PVX CP subunits possess some fixed tertiary structure but this structure is highly unstable and is completely disrupted at temperatures as low as 35 degrees C. The free PVX CP tertiary structure was also disrupted by very low sodium dodecylsulfate and cetyltrimetylammonium bromide concentrations: 3 to 5 moleculs of the surfactants per the CP molecule were sufficient to induce its total disruption. At the same time, these treatments did not result in any changes in the PVX CP secondary structure. Incorporation of the CP subunits into the PVX virions resulted in a strong increase in their stability to effects of increased temperatures and surfactants. This combination of highly labile tertiary structure and rather stable secondary structure of free PVX CP subunits may represent a structural basis for recently observed capacity of the PVX CP moleculs to assume two different functional states in the virion.

[Modified model of potato virus X coat protein structure].

We propose the modified model of the structure of coat protein (CP) subunits in filamentous virions of potato virus X (PVX). The model is similar to the one proposed by us in 2001 for the CP of another helical plant virus (potato virus A) belonging to other (potyvirus) group. In this model the PVX CP molecule consist of two main domains--a bundle of four alpha-helices located close to the virion long axis and a so-called RNP-fold (or abCd-fold) located near the virion surface. Basing on this model we suggest possible mechanism of described by J.G. Atabekov and colleagues structural transition ("remodeling") of the PVX virions resulting from their interaction with virus-specific TGB-1 protein.

[Determination of concentration and aggregate size in influenza virus preparations using the true UV-absorption spectra].

It is well-known that influenza virus (IV) preparations are characterized by very large contribution of light-scattering to their UV absorption spectra. With the help of so called extrapolation method we managed to measure true absorption spectra of IV preparations and to determine absorption coefficients (E0.1(1) (cm, 280)) for the intact IV virions and for IV subviral particles. These coefficients turned out to equal 1.26 +/- 0.17 and 0.96 +/- 0.11 for the virions and subviral particles respectively. The knowledge of exact IV concentration is necessary for quantitative physico-chemical studies of IV virions and their components. It is also shown that UV absorption spectra measurements allow to register IV virion aggregation. Aggregation properties of IV subviral particles were also studied.

[Investigation of helical plant virus ribonucleoprotein structures with the help of tritium planigraphy and theoretical modeling]. / Issledovanie struktury ribonukleotidov spiral'nykh virusov rastenii metodami tritievoi planigrafii i teoreticheskogo modelirovaniia.

The results of the studies of helical plant virus structures by tritium planigraphy (TP) method are discussed. TP method is based on bombardment of macromolecular objects with a stream of tritium atoms, followed by analysis of tritium label distribution along the macromolecule. By combining the TP data with the results of theoretical predictions of the protein structure, it turned out to be possible to propose a model of the coat protein structure in the virions of potato virus X (the type member of potexvirus group) and potato virus A (one of the members of potyvirus group). With the help of TP it also managed to find subtle differences in the coat protein structure between wildtype tobacco mosaic virus (strain U1) and its mutant with two amino acid substitutions in the coat protein and alter host specificity.

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability.

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.

Rate of tetracycline photolysis during irradiation at 365 nm.

Kinetics of photolysis of the antibiotic tetracycline (TC) during irradiation at 365 nm was studied in three buffer solutions usually used for studies on TC binding to its main cell targets--a transcriptional repressor protein TetR and to the ribosome. These buffer solutions contain magnesium ions and an antioxidant--mercaptoethanol or dithiothreitol. The rate of TC photolysis was maximal in medium which contained 14 mM mercaptoethanol and 5 mM magnesium ions. In the absence of mercaptoethanol the photolysis rate was more than twofold decreased. The rate constants and quantum yields of the photolysis were determined under various conditions.

Kinetics of thermal aggregation of tobacco mosaic virus coat protein.

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52 degrees C in a wide range of protein concentrations, [P]0. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52 degrees C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]0 (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42 degrees C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]0 and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates [A/A(lim); t] (t is time and A(lim) is the limiting value of A at t --> infinity). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.

In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment.

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

[Differences in the spatial structure of an envelope protein from tobacco mosaic virus and its mutant, detected by tritium planigraphy]. / Razlichiia v prostranstvennoi strukture belka obolochki v sostave virusa tabachnoi mozaiki i ego mutatnta, obnaruzhivaemye metodom tritievoi planigrafii.

Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile-21-->Thr and Asp-66-->Gly) in the coat protein (CP) gene and in symptoms produced in infected N' plants. The CP structure in TMV-U1 and ts21-66 virions was probed by tritium planigraphy. Compared with the wild-type CP, labeling of the N-terminal region of mutant CP was half as high and suggested its greater shielding. A role of this CP region in virus interactions with the N' resistance system is discussed.

Macroscopic aggregation of tobacco mosaic virus coat protein.

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry "melting" curves, TMV CP in the form of a trimer-pentamer mixture ("4S-protein") has very low thermal stability, with a transition temperature at about 40 degrees C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial alpha-helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.

Appearance of "beta-like" circular dichroism spectra on protein aggregation that is not accompanied by transition to beta-structure.

CD spectra in the 200 to 250 nm spectral region for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV) coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared. It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a protein with high alpha-helix content, which agrees well with results of X-ray diffraction studies. But in the long helical aggregates (and in the TMV virions) TMV CP gives "beta-like" CD spectra similar to those of many other aggregated proteins. From X-ray diffraction data, it is well known that TMV CP subunits do not change their secondary or tertiary structure on assembly into virions or the helical repolymerized protein. Thus, the change in the shape of 200 to 250 nm CD spectra cannot be employed as the sole criterion of the conversion of a protein to beta-structure in the course of aggregation.

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1).

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

Virus-induced permeability transition in mitochondria.

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant.

The differential scanning calorimetry (DSC) 'melting curves' for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain U1) and for its CP ts mutant ts21-66 were measured. Strain U1 and ts21-66 mutant (two amino acid substitutions in CP: 121 --> T and D66 --> G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21-66 at pH 8.0, in the form of small (3-4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72 degrees C for U1 and from 38 to 72 degrees C for ts21-66 CP. In the RNA-free helical virus-like protein assemblies U1 and ts21-66 CP subunits had a thermal stability intermediate between those in 3-4S aggregates and in the virions. ts21-66 helical protein displayed a somewhat lower thermal stability than U1.

Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant.

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 A from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21=>T and D66=>G, both in the 70-A region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21-66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-A region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.