Loren A. Day

Hydrodynamic properties and structure of fd virus

Abstract A length of 8950 ± 200 A and a diameter of 90 ± 10 A have been obtained for fd virus from a simultaneous solution of the Broersma equations relating the length and diameter of a rod-like particle to its rotational, DR, and translational, DT, diffusion coefficients. Measurements of DR were by transient electric birefringence, and of DT by low-angle intensity fluctuation spectroscopy. A mass of (16.4 ± 0.6) × 106 daltons was calculated from the Svedberg equation using our measured values of DT, the sedimentation coefficient and the density increment. These results, together with the molecular weight of fd DNA, give a total number of major coat protein subunits of 2710 ± 110 and a ratio of nucleotides to protein subunits which is definitely non-integral, 2.30 ± 0.11. These measurements help delineate significant structural differences between fd and other filamentous viruses. Also included in this paper is an Appendix (by L. A. Day & S. A. Berkowitz) concerning the number of nucleotides, 6370 ± 140, and the density and refractive index increments of fd DNA.

Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf: Investigation by laser raman spectroscopy

The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy. By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf. The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains. The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both. The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses. The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions. Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus.

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of ≈7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, 〈S〉, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The large-amplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA–protein interface within a virus.

Mass, length, composition and structure of the filamentous bacterial virus fd☆

Determinations by three independent methods gave an average of (14.6 ± 0.6) × 106 daltons for the anhydrous mass of the filamentous bacterial virus fd; a determination of the mass per unit length by light scattering of the virus in solution gave 1560 ±60 daltons/A; and three independent methods show that 12.0±O.2% of the virus mass is from the single-stranded circular DNA molecule. The data give an average axial distance of 3.82 ±0.15 A between major coat protein subunits (5240 daltons each) for virus in solution. The DNA has an up strand and a down strand within the filament, and an average axial distance of 3.29 ± 0.14 A between neighbouring nucleotides in a given strand is obtained from the data. There are 2.32 ±0.07 nucleotides per major coat protein subunit and hence each of the nucleotides cannot interact in the same way with subunits of coat protein. The results provide a basis for the interpretation of X-ray diffraction patterns of oriented fibers of the virus. The uncertainties cited above are 95% confidence limits.

The molecular weight of bacteriophage θ6 and its nucleocapsid

Abstract A diameter of 82 nm, a molecular weight of 99 × 106, and a refractive index increment of 0.152 g−1cm3 were obtained for θ6 bacteriophage from turbidity measurements made with an analytical ultracentrifuge and a conventional spectrophotometer. Turbidity was also used to obtain a diameter of 64 nm and a molecular weight of 40 × 106 for the θ6 nucleocapsid produced when virus solutions are made 2% Triton X-100. The RNA content found for the intact virus by the orcinol method was 10%- by weight, which corresponds to 10 million for the total molecular weight of the three double-stranded RNA molecules comprising the genome. The phospholipid content of the intact virus was found to be 20% by weight. The relative amounts of individual protein species were determined by gel electrophoretic analyses of radioactive virus. The results yielded estimates of the number of copies of each protein in the virion.

Raman optical activity instrument for studies of biopolymer structure and dynamics

The latest version of a multi-channel Raman optical activity (ROA) instrument implementing incident circular polarization (ICP) modulation within a in a backscattering geometry and optimized for measurements on biopolymers in aqueous solution is described. It is based on a fast single-stage, imaging (stigmatic) monochromator equipped with a high optical density holographic notch filter, a holographic transmission grating and a thinned back-illuminated thermoelectrically cooled charge-coupled device (CCD) detector with a high quantum efficiency. A large-aperture longitudinal electro-optic modulator (Pockels cell) is employed to switch between orthogonal circular polarization states in the incident laser radiation. A thick Lyot depolarizer is used for depolarization of the backscattered Raman radiation. This backscattering ICP CCD ROA design is currently realized with two prototype instruments dedicated to studies of the structure and dynamics of biopolymers in aqueous solution. Backscattered ICP ROA spectra of the following samples are presented as typical examples of the excellent performance characteristics: poly(L-glutamic acid) in α-helical and disordered conformations; bovine α-lactalbumin in native and acid molten globule states; human immunoglobulin; calf thymus DNA and both magnesium-bound and magnesium-free phenylalanine-specific transfer RNA; and filamentous bacterial viruses Pf1 and M13. Copyright

Different arrangements of protein subunits and single-stranded circular DNA in the filamentous bacterial viruses fd and Pf1

Abstract A single-stranded circular DNA molecule of 6690 ± 450 nucleotides accounts for 5.5 ± 0.3% of the mass of Pf1 virus. The remaining mass is contributed almost entirely by subunits of the major coat protein. A non-integral nucleotide to subunit ratio of 0.87 ± 0.05 is calculated from the DNA content, the average nucleotide mass (309), and the known mass of one protein subunit (4609). There are therefore 7690 ± 680 major coat protein subunits in the virus. The virus length determined by electron microscopy is 1960 ± 70 nm. The data give an average axial distance of 2.55 ± 0.24 A between protein subunits in dry virus. Since there is an up strand and a down strand of the circular DNA within the virus filament, an axial distance between bases in a given strand of 5.9 ± 0.5 A is calculated. Available X-ray data show that an axial repeat of 72 A, or slightly less, would be expected for dry Pf1 virus (0% relative humidity). A structural model in which 27 protein subunits and 24 nucleotides are contained in this repeat would be consistent with our data. The DNA conformation and the subunit packing in Pf1 differ considerably from those in fd, even though both are filamentous viruses containing single-stranded circular DNA. The uncertainties cited are 95% confidence limits.

Different packaging of DNA in the filamentous viruses Pf1 and Xf

Abstract Xf Virus DNA, like Pf1 DNA, is a single-stranded circular molecule and contains, within experimental error, the same number of nucleotides, 7400. This was unexpected since Pf1 virus is 2 μm long while Xf virus is only 1 μm long. The ratio of nucleotides to major coat protein subunits has been found to be nearly unity in Pf1 and nearly two in Xf, but it is not certain that the ratios have exactly integer values. Calculations give the average axial internucleotide separation in Pf1virus as 5.3 A whereas in Xf virus, the calculated separation is only 2.6 A. The protein subunits in both Pf1 and Xf have calculated axial separations close to 2.6 A. The results provide a solution to a problem encountered in the interpretation of X-ray diffraction patterns of these viruses concerning the number of protein subunits per helical turn.

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization

Significance This work presents a technique for dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) solid-state NMR studies of complex proteins and biological assemblies. The sequential side-chain correlation approach streamlines the site-specific assignment of NMR peaks in multidimensional spectra, a critical step in determining structural information such as distances. When combined with DNP enhancement, fast MAS, and nonuniform sampling, this technique allows for faster data acquisition than previously possible. Applied to the intact Pf1 bacteriophage, sequential side-chain correlation spectra have enabled a virtually complete assignment using DNP data alone. These assignments shed insight into the chemical shift and linewidth changes associated with cryogenic temperatures. Our data point to hydration as a key variable influencing these parameters. An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.