Preparative isolation of basic structural proteins of the influenza virus]

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.     

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.     

Studying Liposomes by Tritium Bombardment

Bilayer liposomes from a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPC:DPPE=8:2, molar ratio) or DPPC labeled with ¹⁴C-DPPC (DPPC:¹⁴C-DPPC) were bombarded with thermally activated tritium atoms. The tritiated liposomes were hydrolyzed by phospholipase C, and the tritium incorporation into different parts of the bilayer along its thickness was determined. The tritium flux attenuation coefficients were calculated for the headgroup (k₁=0.176±0.032 Å^–1) and acylglycerol residue (k₂=0.046±0.004 Å^–1) layers indicating a preferential attenuation of the tritium flux in the headgroup region and relative transparence of the membrane hydrophobic part. The finding is potentially important to apply tritium bombardment for investigation of spatial organization of transmembrane proteins in their native lipid environment.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.     

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.     

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20–30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.     

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B_1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.     

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.     

Investigation of the complex antibiotic INA-5812

A concentrate with the antimicrobial activity has been isolated from the culture broth of Streptomyces roseoflavus INA-Ac-5812. Its further fractionation by reversed-phase HPLC has resulted in six fractions. It has been established by MALDI-TOF and ESI-MSⁿ precision mass-spectrometry methods that the main components of the complex antibiotic are several closely related compounds, presumably of a glycopeptide nature. The fraction containing an individual component with a mass of 1845.788 Da has been characterized by UV/Vis absorbance and fluorescence spectra, amino acid analysis, and derivatization with tris(2,6-dimethoxyphenyl)methyl cation. The activity of fractions against pathogenic microbes has been studied. The results allow the supposition that the INA-5812 antibiotic complex is a glyco- or lipoglycopeptide antibiotic of a new type, which is very promising for further study.