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

Abstract

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.

Analysis of 31P MAS NMR spectra and transversal relaxation of bacteriophage M13 and tobacco mosaic virus

“…M13 and TMV were grown, purified, and concentrated as described previously (Magusin and Hemminga, 1993a). NMR spectra were recorded on a Bruker AM500 and a CXP300 spectrometer operating at a 31p frequency of 202.5 and 121.5 MHz, respectively.…”

“…In simulations for M13 it was assumed that a random distribution of chemical shift tensor orientations exists within the phage. For TMV the orientation of 31P chemical shift tensor for the three different binding sites with respect to the rod-shaped TMV was approximately derived from the 31P atomic coordinates as described earlier (Magusin and Hemminga, 1993a). The best fits to the experimental sideband patterns and lineshapes of M13 and TMV were found by least square fitting, allowing height, baseline, and isotropic shift of simulated sideband patterns and lineshapes to vary.…”

“…Because phosphorus nuclei in M13 and TMV are only present in the phosphodiesters of the encapsulated nucleic acid molecules, 31P NMR spectroscopy can be used to study structure and dynamics of the nucleic acid backbone selectively. Motional lineshape narrowing and transversal relaxation observed in 31P NMR studies of virus gels can neither be explained by (restricted) overall rotation of the virus particles as rigid rods about their length axis, nor by isotropic motion of the phosphodiesters inside (Magusin and Hemminga, 1993a). Instead, a combined diffusion model with fast restricted motions of the encapsulated nucleic acid dominating the spectrum superimposed on slow overall rotation of the virions affecting transversal relaxation provides a consistent picture.…”

“…Sideband intensities 31P in various nucleic acids, generally occur within the ranges -105 ± 10 ppm and -25 + 2 ppm, respectively (Magusin and Hemminga, 1993a;Hemminga et al, 1987), indicating that conformationally induced changes of Fo and F2 are relatively small. For MAS at an angular frequency (Or, the angles C and ( in Eq.…”

“…Similar to TSOR, TcSA is inversely proportional to the square of the resonance frequency w.. In our previous analysis of transversal 31P relaxation of nonspinning samples (Magusin and Hemminga, 1993a), we have assumed that TCSA >> TSOR so that it may be neglected. In contrast, it will be shown in this paper that TSOR is negligible at spinning rates of 4 kHz or higher.…”