Hydrodynamic determination of molecular weight, dimensions, and structural parameters of Pf3 virus

Newman, Jay; Day, Loren A.; Dalack, Gregory W.; Eden, Don

doi:10.1021/bi00257a016

Cited by 23 publications

(15 citation statements)

References 24 publications

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“…This band was assigned as a minor coat protein component of the virion. From the relative intensities it was surmised that the major coat protein constitutes more than 95% of the total protein in the virus, and, according to molecular weight studies, there are approximately 2,500 subunits per virion (3,4,14). The ss DNAbinding protein-DNA complexes, isolated from P13-infected cells, gave a band at -6,000 Mr when disrupted and electrophoresed under the same conditions (13).…”

Section: Resultsmentioning

confidence: 99%

“…On average there are 2.4 nucleotides present in the virion per subunit (4,39), yet there are only two basic amino acids in the subunit. This situation is unique to P13 virus because the major coat proteins of the other viruses have more basic residues than their equivalent number of nucleotides in the virion.…”

Section: Resultsmentioning

confidence: 99%

“…Pf3 bacteriophage and its host P. aeruginosa (PAO1) bearing the RP1 plasmid (1) were obtained originally from D. E. Bradley. Phage Pf3 was propagated and purified from single-plaque isolates (3,4). A standard medium containing 10 g of tryptone, 1 g of yeast extract, 8 g of NaCl, 1 g of glucose, and 220 mg of CaCl2 per liter was used.…”

Section: Methodsmentioning

confidence: 99%

“…These viruses each contain a single-stranded circular DNA molecule encased in a slender protein sheath composed of thousands of identical protein subunits of molecular weight about 5,000 (the major coat protein) and a few copies each of a few coat proteins located at the ends of the virions (the minor coat proteins). Various comparative physical studies have shown that the packing of the DNA inside the different viruses is not the same, and also that intracellular complexes between the DNAs and their single-stranded DNA (ss DNA)-binding proteins are different (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13).…”

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confidence: 99%

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Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

Putterman¹,

Casadevall²,

Boyle³

et al. 1984

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

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The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, fl) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNAbinding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses.Bacteriophage Pf3 infects Pseudomonas aeruginosa PAO1 bearing the RP1 plasmid (1). It is one of a variety of filamentous viruses of Gram-negative bacteria, the best known of which are the closely related fd, fl, and M13 phages, all of which infect male strains of Escherichia coli. Other filamentous phages are Pfl (P. aeruginosa, strain K), Xf (Xanthomonas oryzae), and IKe and Ifl, both of which grow on E. coli strains bearing N and I plasmids, respectively. These viruses each contain a single-stranded circular DNA molecule encased in a slender protein sheath composed of thousands of identical protein subunits of molecular weight about 5,000 (the major coat protein) and a few copies each of a few coat proteins located at the ends of the virions (the minor coat proteins). Various comparative physical studies have shown that the packing of the DNA inside the different viruses is not the same, and also that intracellular complexes between the DNAs and their single-stranded DNA (ss DNA)-binding proteins are different (2-13).The best-characterized of these systems is that of fd (fl, M13), and it serves as the basis for comparisons (see ref. 12 for reviews). Of particular relevance to the present study is that the major coat protein subunits offd span the membrane of the infected. cell before they form a coat around the viral DNA. Their insertion into the membrane involves the processing of a signal sequence from a precursor protein. Dur-ing virus assembly, which occurs at the membrane, the viral DNA leaves its complex with its single-stranded DNA-binding protein as it becomes covered by major coat protein subunits and is extruded into the medium. The assembly and extrusion process does not cause cell lysis.In this paper, we present the results of an investigation of a region on the genome of Pf3 that encodes the DNA-binding protein and the coat protein of this virus. Comparisons of this region of DNA in Pf3 with its fd counterpart indicate close similarities in overall organization yet quite different amino acid sequences for the structural proteins themselves and the absence of a signal sequence for the major coat protein. MATERIALS AND METHODSBacteria and Bacteriophage. Pf3 bacteriophage and its host P. aeruginosa (PAO1) beari...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

Putterman¹,

Casadevall²,

Boyle³

et al. 1984

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

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“…Static and dynamic light scattering measurements were done using a home-made optical arrangement described previously [37], a Lexel argon ion laser operating in TEM 00 mode at 514.5 nm and a Brookhaven BI-9000 correlator. A series of sequential two-minute experiments at a 90° scattering angle of both the average scattered intensity and the intensity autocorrelation function were made after insulin samples were placed in the thermal optical bath and quenched to the measurement temperature.…”

Section: Methodsmentioning

confidence: 99%

Inhibitory Effects of Arginine on the Aggregation of Bovine Insulin

Varughese

Newman

2012

Journal of Biophysics

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Static and dynamic light scattering were used to investigate the effects of L-arginine, commonly used to inhibit protein aggregation, on the initial aggregation kinetics of solutions of bovine insulin in 20% acetic acid and 0.1 M NaCl as a model system for amyloidosis. Measurements were made as a function of insulin concentration (0.5–2.0 mM), quench temperature (60–85°C), and arginine concentration (10–500 mM). Aggregation kinetics under all conditions had a lag phase, whose duration decreased with increasing temperature and with increasing insulin concentration but which increased by up to a factor of 8 with increasing added arginine. Further, the initial growth rate after the lag phase also slowed by up to a factor of about 20 in the presence of increasing concentrations of arginine. From the temperature dependence of the lag phase duration, we find that the nucleation activation energy doubles from 17 ± 5 to 36 ± 3 kcal/mol in the presence of 500 mM arginine.

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Total intensity light scattering from short, optically anisotropic wormlike chains

Allison

1983

Biopolymers

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SynopsisSimple exact equations are derived for intensity light scattering from optically anisotropic wormlike chains in the absence of excluded volume. The results are valid at low scattering angles ( q 2 ( R % ) 5 1) for all wormlike chains from rigid rods to random coils. The present work and an earlier theory [Nagai, K. (1972) Polym. J. 3,67-831 appear to be equivalent, although they were both derived using different methods. The present work is primarily concerned with short wormlike chains, since intensity light scattering from short fragments may provide valuable information about DNA flexibility. By using the results of this work to reanalyze some older light-scattering studies [Godfrey, J. E. & Eisenberg, H. (1976) Biophys.Chem. 5,301-3181, it is shown that anisotropy corrections to polarized light-scattering measurements have been overcorrected in the past. It can be anticipated that future light-scattering experiments will determine the base-pair anisotropy. INTRODUCTIONIn the last 10 years, advances in molecular biology have made it possible to obtain relatively large quantities of monodisperse DNA restriction fragments covering a broad range of molecular weights. The solution configuration of these fragments thus covers the spectrum from rigid rod to random coil. The primary purpose of this article is to examine in some detail the theory of (Rayleigh-Debye) light scattering from anisotropic wormlike chains, particularly short ones on the order of a persistence length long. This model may also be of use, for example, in studying certain viruses or stiff synthetic polymers.The usefulness of light-scattering methods as a tool to extract information on polymer chains in solution was recognized many years ago by Debye.' He determined the integrated intensity for linear chains by modeling the polymer as a large number of optically isotropic point scatterers that are distributed statistically as a Gaussian. More realistic models for stiff linear chains came later. Hermans and Ullman2 incorporated stiffness and connectivity when they considered the light scattered from a wormlike or K r a t k y -P~r o d~-~ chain made up of isotropic point scatterers. Riopolymers, Vol. 22, 1545Vol. 22, -1569Vol. 22, (1983 More recent work has determined integrated intensities for wormlike chains both with and without inclusion of excluded-volume effects.6-8In one important respect, these theories are unrealistic. Units of a real chain are usually optically anisotropic, and this anisotropy gives rise to depolarized light scattering. In general, the polarizability, which is related to the ability of the electrons of a molecule to become displaced under the influence of an electric field, is a symmetrical second-rank tensor.9 Integrated light-scattering intensities for thin rigid rods1°-12 and cylindrically symmetric particlesI3 have been determined for the more general anisotropic case. For certain flexible molecules, light-scattering measurements give unrealistic (even negative) values of RG based on isotropic models, and ...

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Hydrodynamic determination of molecular weight, dimensions, and structural parameters of Pf3 virus

Cited by 23 publications

References 24 publications

Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

Inhibitory Effects of Arginine on the Aggregation of Bovine Insulin

Total intensity light scattering from short, optically anisotropic wormlike chains

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