Modeling of protein spatial structure using tritium planigraphy

The results of protein spatial structure modeling using the tritium planigraphy technique are presented. The knowledge of 3D structure of macromolecules is obligatory for understanding the basic mechanisms of interaction in biological systems and complex technological processes. Known limitations of the X-ray analysis (crystal state) and NMR (molecular weight) make it necessary to seek new approaches to modeling the spatial structure of proteins. Semiempirical tritium planigraphy is one of these approaches. The method is based on bombardment of the object with a beam of hot tritium atoms (E _at ≥ 0.3 eV) and computer simulation. On the example of proteins of different structural classes, we show that this integrated approach can yield a 3D model well consistent with the X-ray data. An important factor is the sequence of searching for contacts between secondary structure elements: the best fit with the native structure is achieved by assembling the elements from the N- to the C-terminus of the polypeptide chain.     

Structures of Helical Plant Virus Ribonucleoproteins as Assessed by Tritium Planigraphy and Theoretical Modeling

This review considers the results of probing the structure of ribonucleoprotein particles of helical plant viruses by tritium planigraphy (TP). This method works by exposing macromolecular targets to a beam of tritium atoms and analyzing the tritium label distribution along the macromolecule length. The TP data combined with theoretical predictions made it possible to propose a structural model of the coat protein for the virions of potato viruses X (the type representative of potexviruses) and A (a potyvirus), which eluded X-ray diffraction analysis so far. TP revealed fine structural differences between the wild-type tobacco mosaic virus (strain U1) and its temperature-sensitive mutant with an altered coat protein and host specificity. The possibilities of using TP for studying the RNA–protein interactions in helical virus particles are discussed.     

Investigation of helical plant virus ribonucleoprotein structures with the help of tritium planigraphy and theoretical modeling

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.     

Differences in spatial structures of the influenza virus M1 protein in crystal, solution and virion

Spatial structure of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) M1 protein in a solution and composition of the virion was studied by tritium planigraphy technique. The special algorithm for modeling of the spatial structure is used to simulate the experiment, as well as a set of algorithms predicting secondary structure and disordered regions in proteins. Tertiary structures were refined using the program Rosetta. To compare the structures in solution and in virion, also used the X-ray diffraction data for NM-domain. The main difference between protein structure in solution and crystal is observed in the contact region of N- and M-domains, which are more densely packed in the crystalline state. Locations include the maximum label is almost identical to the unstructured regions of proteins predicted by bioinformatics analysis. These areas are concentrated in the C-domain and in the loop regions between the M-, N-, and C-domains. Analytical centrifugation and dynamic laser light scattering confirm data of tritium planigraphy. Anomalous hydrodynamic size, and low structuring of the M1 protein in solution were found. The multifunctionality of protein in the cell appears to be associated with its plastic tertiary structure, which provides at the expense of unstructured regions of contact with various molecules-partners.     

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.     

Possibility to study cell membrane topography using tritium planigraphy

The method of tritium planigraphy was adopted for the investigation of intact cells. Conditions for the incorporation of thermally activated tritium atoms in the erythrocytes are described. The accessibility of erythrocytes hemoglobin for tritium was compared to that of free hemoglobin. By comparing specific radioactivities of amino acids it was shown that the incorporation of the label into free hemoglobin was over 100 times higher than into that in erythrocytes. The cell membrane was highly tritiated. Thus the plasma membrane protects the cell inner regions from penetration of the hot tritium atoms. Tritium planigraphy can be used for studying the cell surface topography.     

Modelling protein three-dimensional structure using tritium planigraphy

We propose the use of data on the topography of the label-accessible surface of a protein molecule obtained by the method of tritium planigraphy as a criterion for choosing the optimal intermediate arrangements of alpha-helices in globular proteins so as to model their three-dimensional structures. This approach has been used for modelling the three-dimensional structure of parvalbumin III from pike. The proposed model has been compared with high-resolution X-ray structural data for a related protein, paryvalbumin from carp. The possibilities and limitations of this approach are discussed.     

Intrinsically unstructured regions in the C domain of the influenza virus M1 protein

The M1 matrix protein of the influenza virus is one of the main structural components of the virion that performs several different functions in the infected cell. X-ray analysis (with 2.08 Å resolution) has been performed for the N-terminal part of the M1 protein (residues 2–158) but not for its C-terminal domain (159–252). In the present study, we analyzed the structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (H1N1) strain in acidic solution using tritium planigraphy. The incorporation of tritium label into the domains of the M1 protein were studied; the C domain and the interdomain loops are preferentially accessible to tritium. Analytical centrifugation and dynamic laser light scattering demonstrated anomalous hydrodynamic parameters and low structuredness of the M1 protein, which has also been confirmed by circular dichroism data. Bioinformatic analysis of the M1 protein sequence revealed intrinsically unstructured segments that were concentrated in the C domain and interdomain loops between the N-, M-, and C domains. We suggest that the multifunctionality of the M1 protein in a cell is determined by the plasticity of its tertiary structure, which is caused by the presence of intrinsically unstructured segments.     

Differences in the spatial structure of an envelope protein from tobacco mosaic virus and its mutant, detected by tritium planigraphy]

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.     

Tritium planigraphy: Differences in the spatial structures of the influenza virus M1 protein in crystal,solution, and virion

The structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) in solution at acidic pH and in the composition of the virion has been studied by the tritium planigraphy method. A model of the spatial structure was constructed using a special algorithm simulating the experiment and a set of algorithms for predicting the secondary structure and disordered regions in proteins. The tertiary structure was refined using the Rosetta program. For a comparison of the structures in solution and inside the virion, the data of X-ray diffraction analysis for the NM domain were also used. The main difference in the structures of the protein in solution and the crystalline state is observed in the region of contact of N and M domains, which in the crystalline state is packed more densely. The regions of the maximum label incorporation almost completely coincide with unstructured regions in the protein that were predicted by the bioinformatics analysis. These regions are concentrated in the C domain and in loop regions between M, N, and C domains. The data were confirmed by analytical centrifugation and dynamic light scattering. Anomalous hydrodynamic dimensions and a low structuration of the M1 protein in solution were found. The polyfunctionality of the protein in the cell is probably related to its flexible tertiary structure, which, owing to unstructured regions, provides contact with various partner molecules.     

Potato virus X: structure, disassembly and reconstitution

This paper summarizes some structural characteristics of Potato virus X (PVX), the flexuous filamentous plant potexvirus. A model of PVX coat protein (CP) tertiary structure in the virion proposed on the basis of tritium planigraphy combined with predictions of the protein tertiary structure is described. A possible role of glycosylation and phosphorylation in the CP structure and function is discussed. Two forms of PVX virion disassembly are discussed: (i) the virion co-translational disassembly after PVX CP in situ phosphorylation and (ii) disassembly of PVX triggered by different factors after linear destabilization of the virion by binding of the PVX-coded movement protein (TGBp1) to one end of the polar CP-helix. Special emphasis was placed on a translational activation of encapsidated PVX RNA and rapid disassembly of TGBp1-PVX complexes into free RNA and CP. The results of experiments on the PVX CP repolymerization and PVX reconstitution are considered. In particular, the products assembled from PVX RNA, CP and TGBp1 were examined. Single-tailed particles were found with a helical, head-like structure consisting of helically arranged CP subunits located at the 5'-tail of RNA; the TGBp1 was bound to the end of the head. Translatable 'RNA-CP-TGBp1' complexes may represent the transport form of the PVX infection.     

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.     

A model for the study of the mechanism of a low pH-induced interaction of the virus fusion proteins and cell membranes

A model is proposed for the study of molecular mechanisms of a low pH-induced interaction of fusion proteins of enveloped viruses and cell membranes. The model consists of large monolamellar liposomes containing ionophore nigericin in their membranes and ectodomains of fusion protein in their inner space. The process of interaction of the protein with the lipid bilayer is triggered by acidification of the liposomal constituents to the pH of fusion with the help of nigericin by adding citric acid to the outer medium. To visualize the protein structural reorganization, the tritium planigraphy was used.Comparison of the values of specific labelling of the proteins and distribution of radioactivity in individual amino acids in control (at neutral pH) and experimental liposome samples (at the pH of fusion) permits to realise the character of protein-membrane interaction. We have obtained the first results in the study of interaction of the bromelain-released soluble ectodomain of the HA^XX molecule (BHA)—with the lipid membrane. The observed increase in the protein specific activity and selective increase in the specific activity of hydrophobic amino acids Ile, Phe and Tyr in experimental liposome samples as compared with the controls did not contradict to the conventional concept, that a hydrophobic N-terminus of HA2 subunit of hemagglutinin is responsible for its interaction with lipid membranes.     

The Spatial Arrangement of the Wild-Type and Mutant Tobacco Mosaic Virus Coat Proteins Assessed by Tritium Planigraphy

Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile21 Thr and Asp66 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. The role of this CP region in virus interactions with the N" resistance system 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.     

Calcium-binding protein in sieve tube exudate

A calcium-binding macromolecule, with an estimated molecular weight greater than 100,000, was detected in phloem exudate from Cucurbita maxima and related species. The macromolecule was a component of sieve tube sap, rather than a contaminant leached from cell walls or cut parenchyma cells during exudate collection. The protein nature of this macromolecule was deduced from its size, lability, susceptibility to proteolytic digestion, and by the dependence of calcium-binding activity on thiol-protecting agents. This protein is distinct from the major proteins of exudate and does not appear to be related to calmodulin.Abbreviations SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - CBP calcium-binding protein     

An estimation of the dimensions of the phycocyanin molecule

Knowledge of the total particle volume and the specific volumes of the constituent polar and nonpolar amino acid residues of a globular protein may be used in suitable instances to estimate the size and shape, of the macromolecule. Use has been made of the data, which is available in the literature for phycocyanin from Plectonema calothricoidcs, to predict possible models for the monomer and hexamer forms of this protein. The monomer is well represented by a prolate ellipsoid with semiaxes of 45 and 17Å and an approximate molecular weight of (46000). The hexamer is envisaged as the aggregate of six such monomers, in juxtaposition, with their major axes parallel so as to form a closed ring structure of about 268000 molecular weight. These proposed models are consistent with the previously published electron micrographs and hydrodynamic properties of this protein.     

Conformational Differences between Native and Recombinant Horseradish Peroxidase Revealed by Tritium Planigraphy

Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed. An unexpected result of the study is a finding on high accessibility of a catalytic histidine residue in solution. An effect of low dose (3 Gy) of irradiation on the accessibility of amino acid residues has been unequivocally demonstrated. The data can be interpreted as swelling of the compact folding and increase in the surface hydrophilicity of the recombinant enzyme. In the case of native enzyme, irradiation does not cause remarkable changes in the accessibility of amino acid residues indicating the possible extensive radical modification of the native enzyme in the life-course of the cell. The catalytic histidine is an exception. It becomes inaccessible after the enzyme irradiation, while its accessibility in the recombinant enzyme increases. An additional observation of a 5-fold decrease in the rate constant towards hydrogen peroxide points to the destructive effect of irradiation on the hydrogen bond network in the distal domain of the native enzyme molecule and partial collapse of the active site pocket.     

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.     

ALGAE AS USEFUL TOOLS IN CELLULOSE RESEARCH

This presentation will review the 1976 discovery of the enzyme complex in cellulose biosynthesis in Oocystis apiculata. A linear terminal complex (TC) was found to be associated with a microfibril, and from other freeze fracture applications, TCs have been found in many different algal genera. In fact, the algae have the most diverse and complex TCs among all organisms. TC diversity in terms of the evolution of cellulose biogenesis will be discussed. Combining the latest information from biochemistry and molecular genetics, the multiplicity of cellulose biogenesis will be reviewed. Cellulose molecular weight, crystalline structure, and mode of glycosylation for polymer formation all indicate that cellulose biogenesis is an extremely complex process. Major questions still remain, and the enzymes for cellulose biosynthesis have yet to be crystallized and their structure elucidated; however, the wealth of new information on cellulose structure and biosynthesis from algae to vascular plants, including bacteria and tunicates, all point to a very exciting and useful area of research. The algae have played key roles in our understanding of nature's most abundant macromolecule.