Analysis of 31P nuclear magnetic resonance lineshapes and transversal relaxation of bacteriophage M13 and tobacco mosaic virus.

The experimentally observed 31P lineshapes and transversal relaxation of 15% (wt/wt) M13, 30% M13, and 30% tobacco mosaic virus (TMV) are compared with lineshapes and relaxation curves that are simulated for various types of rotational diffusion using the models discussed previously (Magusin, P. C. M. M., and M. A. Hemminga. 1993. Biophys. J. 64:1851-1860). It is found that isotropic diffusion cannot explain the observed lineshape effects. A rigid rod diffusion model is only successful in describing the experimental data obtained for 15% M13. For 30% M13 the experimental lineshape and relaxation curve cannot be interpreted consistently and the TMV lineshape cannot even be simulated alone, indicating that the rigid rod diffusion model does not generally apply. A combined diffusion model with fast isolated motions of the encapsulated nucleic acid dominating the lineshape and a slow overall rotation of the virion as a whole, which mainly is reflected in the transversal relaxation, is able to provide a consistent picture for the 15 and 30% M13 samples, but not for TMV. Strongly improved lineshape fits for TMV are obtained assuming that there are three binding sites with different mobilities. The presence of three binding sites is consistent with previous models of TMV. The best lineshapes are simulated for a combination of one mobile and two static sites. Although less markedly, the assumption that two fractions of DNA with different mobilities exist within M13 also improves the simulated lineshapes. The possible existence of two 31P fractions in M13 sheds new light on the nonintegral ratio 2.4:1 between the number of nucleotides and protein coat subunits in the phage: 83% of the viral DNA is less mobile, suggesting that the binding of the DNA molecule to the protein coat actually occurs at the integral ratio of two nucleotides per protein subunit.     

2D exchange 31P NMR spectroscopy of bacteriophage M13 and tobacco mosaic virus.

Two-dimensional (2D) exchange 31P nuclear magnetic resonance spectroscopy is used to study the slow overall motion of the rod-shaped viruses M13 and tobacco mosaic virus in concentrated gels. Even for short mixing times, observed diagonal spectra differ remarkably from projection spectra and one-dimensional spectra. Our model readily explains this to be a consequence of the T2e anisotropy caused by slow overall rotation of the viruses about their length axis. 2D exchange spectra recorded for 30% (w/w) tobacco mosaic virus with mixing times < 1 s do not show any off-diagonal broadening, indicating that its overall motion occurs in the sub-Hz frequency range. In contrast, the exchange spectra obtained for 30% M13 show significant off-diagonal intensity for mixing times of 0.01 s and higher. A log-gaussian distribution around 25 Hz of overall diffusion coefficients mainly spread between 1 and 10(3) Hz faithfully reproduces the 2D exchange spectra of 30% M13 recorded at various mixing times in a consistent way. A small but notable change in diagonal spectra at increasing mixing time is not well accounted for by our model and is probably caused by 31P spin diffusion.     

Ion etching of tobacco mosaic virus and T4 bacteriophage.

A technique for the etching of biological materials by accelerated nitrogen ions is described. Upon exposure to such a beam, tobacco mosaic virus becomes thinner in diameter and the head of T4 becomes smaller. Experiments on radioactively labeled T4 suggest that the particle's protein is more susceptible to removal by the incident ions, and that the residual material is predominantly nucleic acid.     

NMR relaxation processes of 31P in macromolecules

³¹P-Nmr relaxation parameters (spin-lattice relaxation time, linewidth, and nuclear Overhauser effect) were obtained at three different frequencies for poly(U) and a well-defined (145 ± 3 base-pair) fragment of DNA in solution. Data sets for the two samples were analyzed by theories which included relaxation by the mechanisms of ³¹P chemical shift anisotropy as well as by ¹H-³¹P dipole–dipole interaction. Neither data set could be satisfactorily described by a single correlation time. A model of a rigid rotor most nearly fits the data for the DNA molecule. Parameters obtained from the least-square fit indicate (1) that the DNA undergoes anisotropic reorientation with a correlation time τ₀ = 6.5 × 10^?7 sec for the end-to-end motion, (2) the ratio of diffusion constants D_∥/D_⊥ is 91, and (3) that the linewidth is due to chemical shift dispersion to the extent of 0.5 ppm. Some deviations of the calculated from the observed values suggested that significant torsional and bending motions may also take place for this DNA. Another model which contains isotropic motion but with a broad distribution of correlation times was required to fit the data for poly(U). A log ? χ² distribution function of correlation times [Scheafer, J. (1973) Macromolecules 6 , 881–888] described well the motion of poly(U) with the average correlation time τ = 3.3 × 10^?9 sec and a distribution parameter p = 14.     

Fluoride effects on 31P NMR spectra of macrophages

31P High resolution nuclear magnetic resonance studies have been carried out on the P388D1 tumoral cell line and the BCG elicited alveolar rabbit macrophages both in sedimented cells and in perfused agarose-embedded cells. When the cells were sufficiently oxygenated, the phosphorylated sugars and ATP concentrations attained high levels. The intensity of the peak representing phosphorylated sugars varied inversely with ATP level when macrophagic cells were treated by NaF. The identities of the phosphorylated sugars were revealed by 1H and 31P NMR studies of the P 388D1 cells perchloric extracts.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Flow dichroic spectra of tobacco mosaic virus and their protein assemblies

Ultraviolet flow dichroisms of tobacco mosaic virus (TMV), TMV-RNA and TMV-protein were measured using three strains of TMV. 1. Large positive dichroisms were observed with three strains of TMV, namely ordinary, bean form of bean, and tomato strains (TMV-OM, TMV-B and TMV-T, respectively) at about 255, 276, 284 and 290 nm. The positive dichroisms were confirmed with reconstituted protein assemblies of TMV-OM and TMV-B at about 276, 284 and 290 nm where tyrosine and tryptophan residues of these proteins contribute. These results show that the electronic transition moments of their base groups and aromatic groups are nearly parallel to the polymer axis. It is suggested that there is a strong interaction between the base groups of RNA and aromatic groups of amino acid residues in TMV. 2. A small negative dichroism was observed near 296-300 nm with intact TMV-OM and TMV-T and with the reconstituted protein polymer of TMV-OM. But negative dichroism was not observed either with the intact virus or the protein assembly of TMV-B. 3. Isolated RNA from TMV-OM, TMV-B and TMV-T showed no dichroism. The configuration of RNA in TMV appears to be imposed on it by its packing with the protein.     

Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.     

Tendencies of 31P chemical shifts changes in NMR spectra of nucleotide derivatives.

31P NMR chemical shifts of the selected mono- and oligonucleotide derivatives, including the compounds with P-N, P-C, P-S bonds and phosphite nucleotide analogues have been presented. The influence of substituents upon 31P chemical shifts has been discussed. The concrete examples of 31P chemical shifts data application in the field of nucleotide chemistry have been considered.     

Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies.

The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.     

Use of 31P and 13C NMR to study enzyme mechanisms

A number of complex biochemical problems have been solved recently by application of new techniques in which 31P and 13C NMR spectroscopy is used. Oxygen isotope exchange phenomena were studied by these NMR methods and used to analyze individual mechanistic events in enzymatic reactions. The existence of intermediates in the reactions catalyzed by glutamine synthetase (EC 6.3.1.2) and carbamyl-phosphate synthetase (EC 2.7.2.9) has been established as well as the kinetic competence of these intermediates for each enzyme. The NMR theory and kinetic experiments required to conduct such studies are discussed.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.