| 1. |
- Ahadi, Aylin, et al.
(författare)
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Three-Dimensional Simulation of Nanoindentation Response of Viral Capsids. Shape and Size Effects
- 2009
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 113:11, s. 3370-3378
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Tidskriftsartikel (refereegranskat)abstract
- The nanoindentation response of empty viral capsids is modeled using three-dimensional finite element analysis. Simulation with two different geometries, spherical and icosahedral, is performed using the finite element code Abaqus. The capsids are modeled as nonlinear Hookean elastic, and both small and large deformation analysis is performed. The Young's modulus is determined by calibrating the force-indentation curve to data from atomic force microscopy (AFM) experiments. Force-indentation curves for three different viral capsids are directly compared to experimental data. Predictions are made for two additional viral capsids. The results from the simulation showed a good agreement with AFM data. The paper demonstrates that over the entire range of virus sizes (or Foppl-von Karman numbers) spherical and icosahedral models yield different force responses. In particular, it is shown that capsids with dominantly spherical shape (for low Foppl-von Karman numbers) exhibit nearly linear relationship between force and indentation, which has been experimentally observed on the viral shell studies so far. However, we predict that capsids with significant faceting (for large Foppl-von Karman numbers) and thus more pronounced icosahedral shape will exhibit rather nonlinear deformation behavior.
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| 2. |
- Castelnovo, M, et al.
(författare)
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Binding effects in multivalent Gibbs-Donnan equilibrium
- 2006
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Ingår i: Europhysics Letters. - : IOP Publishing. - 0295-5075 .- 1286-4854. ; 73:4, s. 635-641
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Tidskriftsartikel (refereegranskat)abstract
- The classical Gibbs-Donnan equilibrium describes excess osmotic pressure associated with confined colloidal charges embedded in an electrolyte solution. In this work, we extend this approach to describe the influence of multivalent ion binding on the equilibrium force acting on a charged rod translocating between two compartments, thereby mimicking ionic effects on force balance during in vitro DNA ejection from bacteriophage. The subtle interplay between Gibbs-Donnan equilibrium and adsorption equilibrium leads to a non-monotonic variation of the ejection force as multivalent salt concentration is increased, in qualitative agreement with experimental observations.
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| 3. |
- Castelnovo, M., et al.
(författare)
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DNA ejection from bacteriophage: Towards a general behavior for osmotic-suppression experiments
- 2007
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Ingår i: European Physical Journal E. Soft Matter. - : Springer Science and Business Media LLC. - 1292-8941 .- 1292-895X. ; 24:1, s. 9-18
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Tidskriftsartikel (refereegranskat)abstract
- We present in this work in vitro measurements of the force ejecting DNA from two distinct bacteriophages (T5 and lambda using the osmotic-suppression technique. Our data are analyzed by revisiting the current theories of DNA packaging in spherical capsids. In particular we show that a simplified analytical model based on bending considerations only is able to account quantitatively for the experimental findings. Physical and biological consequences are discussed.
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| 4. |
- Evilevitch, Alex
(författare)
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Effects of condensing agent and nuclease on the extent of ejection from phage lambda
- 2006
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 110:44, s. 22261-22265
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Tidskriftsartikel (refereegranskat)abstract
- We have recently demonstrated, that DNA ejection from bacteriophage I can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed sand hence for which there is no internal driving force for ejections is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage I incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease ( DNase I) or DNA condensing agent ( spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximate to 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.
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| 5. |
- Evilevitch, Alex, et al.
(författare)
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Effects of salt concentrations and bending energy on the extent of ejection of phage genomes
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 94:3, s. 1110-1120
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Tidskriftsartikel (refereegranskat)abstract
- Recent work has shown that pressures inside dsDNA phage capsids can be as high as many tens of atmospheres; it is this pressure that is responsible for initiation of the delivery of phage genomes to host cells. The forces driving ejection of the genome have been shown to decrease monotonically as ejection proceeds, and hence to be strongly dependent on the genome length. Here we investigate the effects of ambient salts on the pressures inside phage-l, for the cases of mono-, di-, and tetravalent cations, and measure how the extent of ejection against a fixed osmotic pressure (mimicking the bacterial cytoplasm) varies with cation concentration. We find, for example, that the ejection fraction is halved in 30 mM Mg21 and is decreased by a factor of 10 upon addition of 1 mM spermine. These effects are calculated from a simple model of genome packaging, using DNA-DNA repulsion energies as determined independently from x-ray diffraction measurements on bulk DNA solutions. By comparing the measured ejection fractions with values implied from the bulk DNA solution data, we predict that the bending energy makes the d- spacings inside the capsid larger than those for bulk DNA at the same osmotic pressure.
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| 6. |
- Grayson, Paul, et al.
(författare)
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The effect of genome length on ejection forces in bacteriophage lambda.
- 2006
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Ingår i: Virology. - : Elsevier BV. - 1096-0341 .- 0042-6822. ; 348:2, s. 430-436
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Tidskriftsartikel (refereegranskat)abstract
- A variety of viruses tightly pack their genetic material into protein capsids that are barely large enough to enclose the genome. In particular, in bacteriophages, forces as high as 60 pN are encountered during packaging and ejection, produced by DNA bending elasticity and self-interactions. The high forces are believed to be important for the ejection process, though the extent of their involvement is not yet clear. As a result, there is a need for quantitative models and experiments that reveal the nature of the forces relevant to DNA ejection. Here, we report measurements of the ejection forces for two different mutants of bacteriophage λ, λb221cI26 and λcI60, which differ in genome length by 30%. As expected for a force-driven ejection mechanism, the osmotic pressure at which DNA release is completely inhibited varies with the genome length: we find inhibition pressures of 15 atm and 25 atm, for the short and long genomes, respectively, values that are in agreement with our theoretical calculations.
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| 7. |
- Ivanovska, Irena, et al.
(författare)
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Internal DNA pressure modifies stability of WT phage
- 2007
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 104:23, s. 9603-9608
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Tidskriftsartikel (refereegranskat)abstract
- dsDNA in bacteriophages is highly stressed and exerts internal pressures of many atmospheres (1 atm = 101.3 kPa) on the capsid walls. We investigate the correlation between packaged DNA length in A phage (78-100% of WT DNA) and capsid strength by using an atomic force microscope indentation technique. We show that phages with WT DNA are twice as strong as shorter genome mutants, which behave like empty capsids, regardless of high internal pressure. Our analytical model of DNA-filled capsid deformation shows that, because of DNA-hydrating water molecules, an osmotic pressure exists inside capsids that increases exponentially when the packaged DNA density is close to WT phage. This osmotic pressure raises the WT capsid strength and is approximately equal to the maximum breaking force of empty shells. This result suggests that the strength of the shells limits the maximal packaged genome length. Moreover, it implies an evolutionary optimization of WT phages allowing them to survive greater external mechanical stresses in nature.
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| 8. |
- Jeembaeva, Meerim, et al.
(författare)
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Osmotic pressure: resisting or promoting DNA ejection from phage?
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 381:2, s. 310-323
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Tidskriftsartikel (refereegranskat)abstract
- Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.
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| 9. |
- Koester, Sarah, et al.
(författare)
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Influence of Internal Capsid Pressure on Viral Infection by Phage lambda
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 97:6, s. 1525-1529
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Tidskriftsartikel (refereegranskat)abstract
- Ejection of the genome from the virus, phage, is the initial step in the infection of its host bacterium. In vitro, the ejection depends sensitively on internal pressure within the virus capsid; however, the in vivo effect of internal pressure on infection of bacteria is unknown. Here, we use microfluidics to monitor individual cells and determine the temporal distribution of lysis due to infection as the capsid pressure is varied. The lysis probability decreases markedly with decreased capsid pressure. Of interest, the average lysis times remain the same but the distribution is broadened as the pressure is lowered.
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| 10. |
- Lander, Gabriel C., et al.
(författare)
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Bacteriophage lambda stabilization by auxiliary protein gpD: Timing, location, and mechanism of attachment determined by cryo-EM
- 2008
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 16:9, s. 1399-1406
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Tidskriftsartikel (refereegranskat)abstract
- We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-angstrom-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the 14 N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryo-EM density, adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.
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| 11. |
- Löf, David, et al.
(författare)
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Dynamic and Static Light Scattering Analysis of DNA Ejection from Phage λ
- 2007
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Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755. ; E 76:1
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Tidskriftsartikel (refereegranskat)abstract
- With the aid of time-resolved dynamic light scattering (DLS) and static light scattering (SLS), we have analyzed the ejection kinetics from the bacterial virus bacteriophage (or phage) lambda, triggered in vitro by its receptor. We have used DLS to investigate the kinetics in such a system. Furthermore, we have shown that both SLS and DLS can be interchangeably used to study the process of phage DNA release. DLS is superior to SLS in that it also allows the change in the light scattering arising from each of the components in the system to be monitored under conditions such that the relaxation times are separable. With help of these two methods we present a model explaining the reason for the observed decrease in the scattering intensity accompanying DNA ejection from phage. We emphasize that ejection from phage capsid occurs through a very long tail (which is nearly three times longer than the capsid diameter), which significantly separates ejected DNA from the scattering volume of the capsid. The scattering intensity recorded during the DNA ejection process is the result of a change in the form factor of the phage particle, i.e., the change in the interference effects between the phage capsid and the DNA confined in the phage particle. When the DNA molecule is completely ejected it remains in the proximity of the phage for some time, thus contributing to the scattering signal as it diffuses away from the phage capsid, into the scattering volume and returns to its unperturbed chain conformation in bulk solution. The free DNA chain does not contribute to the scattered intensity, when measured at a large angle, due to the DNA form factor and the low concentration. Although the final diffusion-controlled step can lead to overestimation of the real ejection time, we can still use both scattering methods to estimate the initial DNA ejection rates, which are mainly dependent on the pressure-driven DNA ejection from the phage, allowing studies of the effects of various parameters affecting the ejection.
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| 12. |
- Löf, David, et al.
(författare)
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Forces Controlling the Rate of DNA Ejection from Page λ
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 368:1, s. 55-65
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Tidskriftsartikel (refereegranskat)abstract
- The goal of this work was to investigate how internal and external forces acting on DNA affect the rate of genome ejection from bacteriophage λ after the ejection is triggered in vitro by a λ receptor. The rate of ejection was measured with time-resolved static and dynamic light scattering, while varying such parameters as temperature and packaged DNA length, as well as adding DNA-binding proteins to the host solution. We found that temperature has a strong effect on the ejection rate, with an exponential increase of the initial ejection rate as a function of temperature. This can possibly be explained by the temperature-induced conformational changes in the tail pore-forming proteins where the “open” conformation dominates over “closed”, at elevated temperatures. The DNA length also had an effect on initial ejection rate, with a nearly linear dependence comparing the three different genomes (37.7, 45.7 and 48.5 kb DNA), with faster ejection rate for longer genomes. Since the initial rate of ejection increases in an almost direct relationship with the length of the genome, the total time needed to eject DNA completely appeared to be nearly constant for all three DNA length phage mutants. The increased initial rate of ejection with increasing DNA length is due to the increased DNA bending and inter-strand repulsion forces for the longer DNA chains. Finally, we also show that addition of non-specific DNA-binding proteins (HU and DNase I) increases the rate of ejection by exerting additional “pulling” forces on the DNA that is being ejected.
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| 13. |
- Nurmemmedov, Elmar, et al.
(författare)
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Biophysics of viral infectivity: matching genome length with capsid size.
- 2007
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994. ; 40:4, s. 327-356
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Tidskriftsartikel (refereegranskat)abstract
- In this review, we discuss recent advances in biophysical virology, presenting experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded (ds) DNA bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into a viral capsid is highly pressurized, which provides a force for the first step of passive injection of viral DNA into a bacterial cell. Moreover, specific studies on capsid strength show a strong correlation between genome length, and capsid size and robustness. The implications of these newly appreciated physical properties of a viral particle with respect to the infection process are discussed.
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| 14. |
- Roos, W. H., et al.
(författare)
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Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms
- 2007
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 64:12, s. 1484-1497
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Forskningsöversikt (refereegranskat)abstract
- The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular.
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