The use of thermally activated tritium atoms for structural-biological investigations: The topography of the TMV protein-accessible surface of the virus

Goldanskii, V. I.; Kashirin, I. A.; Shishkov, A.V.; Baratova, Ludmila A.; Grebenshchikov, N. I.

doi:10.1016/0022-2836(88)90638-9

Cited by 37 publications

(43 citation statements)

References 15 publications

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“…Amino acid analysis of the labelled peptides was done in a Hitachi 835 analyser according to the method described by Goldanskii et al (1988). Radioactivity of the fractions was counted on a Delta-300 fl-spectrometer (Tracor Analytic) in a dioxane-based liquid scintillation cocktail.…”

Section: Enzymic Hydrolysis Of the Labelled Pvx Proteinmentioning

confidence: 99%

“…Recently, the method of tritium planigraphy has been used to study the label-accessible surface topography and the spacial organization of biological macromolecules and supramolecular systems (Alyonycheva et al, 1987;Filatov et al, 1987;Goldanskii et al, 1988;Jusupov & Spirin, 1986;Neiman et al, 1986). The method is based on the high reactivity of tritium atoms produced by thermal dissociation of tritium molecules on a tungsten filament heated to 2000 K (Goldanskii et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…The method is based on the high reactivity of tritium atoms produced by thermal dissociation of tritium molecules on a tungsten filament heated to 2000 K (Goldanskii et al, 1988). After bombardment of a biological sample with hot tritium atoms the intramolecular distribution of the tritium label is analysed by standard protein chemistry methods.…”

Section: Introductionmentioning

confidence: 99%

“…Of crucial importance is the fact that in addition to the tritium exchange at labile bonds (O-T, S-T and N-T), the exchange also occurs at informational C-H bonds, which are very stable (Goldanskii et al, 1988). This method has been applied recently in the determination of the labelaccessible surface of tobacco mosaic virus (TMV) particles (Goldanskii et al, 1988). The results showed that the method of bombardment with hot tritium atoms might be used to study complex biological entities such as viruses.…”

Section: Introductionmentioning

confidence: 99%

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The topography of the surface of potato virus X: tritium planigraphy and immunological analysis

Baratova

Grebenshchikov

Shishkov

et al. 1992

Journal of General Virology

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Thermally activated tritium atoms were used to probe the surface topography of the coat protein of potato virus X (PVX) potexvirus. The accessibility profile of amino acid residues in the polypeptide chain was determined from data on the intramolecular distribution of tritium label in the PVX coat protein. Tryptic peptides T1 and T2, as well as parts of peptides T3 and T5, from the PVX particles were all located in the Nterminal region of the PVX coat protein and were accessible to tritium labelling, whereas the C-terminal region of the coat protein was practically inaccessible to it. Indirect ELISA and immunoblotting with two PVXspecific monoclonal antibodies confirmed that the N terminus of the coat protein (residues 1 to 56) was exposed on the virus surface, and furthermore that this region forms a highly immunogenic virus-specific antigenic region. The data obtained support the spatial model of PVX, in which the N-terminal amino acids of the coat protein are exposed at the particle surface, and the C-terminal region is buried in the particle. The spatial organization of the PVX coat proteins differs from the model proposed for other filamentous plant viruses such as potyviruses and tobamoviruses where both the N and C termini of the coat protein are located at the particles' surface.

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Section: Enzymic Hydrolysis Of the Labelled Pvx Proteinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The topography of the surface of potato virus X: tritium planigraphy and immunological analysis

Baratova

Grebenshchikov

Shishkov

et al. 1992

Journal of General Virology

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“…ribosomal proteins ͉ ribosome dissociation ͉ ribosomal surface ͉ tritium labeling T he hot tritium bombardment technique is based on replacement of hydrogen by tritium in covalent bonds of thin surface layer in macromolecules (1). The technique appears to be the most direct approach for studies of protein topography on surfaces of biological structures.…”

mentioning

confidence: 99%

A protein residing at the subunit interface of the bacterial ribosome

Agafonov

Kolb

Nazimov

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

104

108

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Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.ribosomal proteins ͉ ribosome dissociation ͉ ribosomal surface ͉ tritium labeling T he hot tritium bombardment technique is based on replacement of hydrogen by tritium in covalent bonds of thin surface layer in macromolecules (1). The technique appears to be the most direct approach for studies of protein topography on surfaces of biological structures. Protein exposure on the surfaces of viruses (1-3), membranes (4), and ribosomes (5-9) has been determined by the use of this technique.Studying proteins of the ribosome surface with this method has lead to the finding that unidentified minor component of the ribosome corresponding to spot Y on the ribosomal protein map becomes highly exposed on dissociation of the 70S ribosome into subunits (5, 7, 9). The reassociation of ribosomal subunits by increasing Mg 2ϩ concentration resulted in reshielding of the protein Y, suggesting its location at the ribosomal subunit interface (9). In this study, the protein was identified and isolated. Its interface location was confirmed by experiments on hot tritium bombardment. The protein was shown to support the 70S ribosome in associated state at low concentration of Mg 2ϩ . Materials and MethodsMaterials. Buffer reagents were obtained from Sigma, acrylamide and methylenebisacrilamide were from Fluka, urea was from Bio-Rad, DNase I was from Serva, Butyl-Toyopearl 650S from Toyo Soda (Tokyo), and DEAE-Sepharose Fast Flow from Pharmacia. Sucrose, acetone, hydrogen peroxide, and acetic acid were from ReaKhim (Moscow, Russia).Preparation of 70S Ribosomes and Total Ribosomal Protein. Ribosomes were prepared from Escherichia coli MRE-600 cells according to Staehelin et al. (10) with modifications described in ref. 9. Salt-washed ribosomes used in binding assay, as well as ribosomal subunits, were prepared as described by Gavrilova et al. (11) with substitution of pelleting by ultracentrifugation for precipitation with (NH 4 ) 2 SO 4 at the final stage of the procedure.The standard procedure of acetic acid extraction and precipitation with acetone (12) was used to p...

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Modelling protein three-dimensional structure using tritium planigraphy

et al. 1991

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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.

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The use of thermally activated tritium atoms for structural-biological investigations: The topography of the TMV protein-accessible surface of the virus

Cited by 37 publications

References 15 publications

The topography of the surface of potato virus X: tritium planigraphy and immunological analysis

The topography of the surface of potato virus X: tritium planigraphy and immunological analysis

A protein residing at the subunit interface of the bacterial ribosome

Modelling protein three-dimensional structure using tritium planigraphy

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