In the second case study, we studied the in vitro lambdain phage DNA extraction process. In the last case study, we studied the DNA extraction process of lambdain phage in vivo.
Introduction
Interestingly, the degree of inactivation was shown to be dependent on the length of the chain. Another case in which bonds have been used to design new proteins is the rewiring of the WASP family of signaling proteins [ 28 ].
Statistical mechanical model of tethered receptor-ligand pairs
The configurations of the ligands in solution and the configurations of the tethers contribute to the degeneracy of each energy level. Binding of free ligand to the receptor has a favorable energy but reduces the entropy of the ligands in solution.
Polymer physics of protein tethers
A comparison between the two polymer models shows that the random walk model is an acceptable approximation of the worm-like chain model for long chains. To find the probability density of the cis-ligand as seen by the receptor, we need to count the tether conformations where the receptor and cis-ligand are close to each other.
Signaling proteins: Roles of tether geometry and length
Simple switches
To find D, we turn to the simple switch titration experiments of Dueber et al. The prediction for the cable length dependence of the simple switch is shown in Figure 2.5.
Complex switches
- Class I switches
- Class II switches
- Applications to reprogrammed N-WASP
- SH3/cis-ligand interactions
The effective concentration of cis-ligand sees the black receptor when the white receptor/cis-ligand pair is unbound. The effective concentration of cis-ligand sees the white receptor when the black receptor/cis-ligand pair is bound.
Discussion
The presence of large macromolecules such as Arp2/3 can affect the conformations available for tethering, leading to a decrease in the effective concentration of cis -ligand. A new round of experiments, similar to the work of Krishnamurthy et al., exploring the thread length dependence of SH3/cis-ligand binding would be useful to explore this issue [11].
Appendix: A kinetic model for the simple switch
First, we find the probability distribution of the distance between the receptor and its cis-ligand. This distribution is then used as an effective entropic potential that controls the dynamics of the cis ligand. The effective concentration is the probability density of the cis ligand in the vicinity of the receptor.
A simulation of the complete kinetic model. a) The probabilities and concentrations forS,SL and ST as a function of time forR=.034 s−1.
Appendix: Polymer models
The wormlike chain
We are interested in the probability that the z component of the end-to-end distance vector takes on a given value. To find the matrix for the exponential matrix of L in this basis, we must first find the matrix forL in the basis L0. We are interested in the case of free edges, so we can integrate over z and z0, which represent the orientations of the final and initial tangent vectors.
We can find the probability distribution for the end-to-end distance vector by noting that it is related to the probability distribution of the z-component of the end-to-end distance vector through the expression .
The freely-jointed chain
If we were interested in the case where all the segments are the same size, then we can get an analytical solution with the help of the contour integral. We can make this integral by closing the contour with an infinite semicircle, provided that the argument of the integral vanishes as the radius of the semicircle goes to infinity. So if x < 0 we close the contour at the bottom and if x > 0 we close it at the top.
This is not surprising because the finite segment length imposes a dependence between the x, y, and z components of R.
Computing probability distributions with delta functions
We can approximate this density by taking the probability that Y is within the window [y−, y+] and dividing by the width of the window, 2. 2.86). Returning to the case of the bound ligand and receptor, we had the probability density functions p1(~x1;L1) and p2(~x2;L2).
Acknowledgements
Furthermore, the internal force of capsid DNA resisting packing increases monotonically with decreasing salt charge. These experiments illustrated the dynamics of the ejection process and provided some insight into the frictional forces experienced by DNA as it exits the capsid [84–86] . The main quantity measured in these experiments is the rate of ejection as a function of the amount of DNA inside the capsid.
Another way to study ejection and packaging is to look at the structure of the DNA itself inside the capsid.
Experimental design
In this work, we focus on measuring the rate and force of DNA ejection in bacteriophage lambda as a function of charge at fixed ionic strength. Therefore, we suggest that the charge of the counterion species is an important control parameter for this system. In these bursts, the first piece of DNA to exit the capsid gets stuck at the origin, leading to a very bright piece of DNA.
The presence of the glucose oxidase/catalase oxygen scavenging system prevents photo damage caused by oxygen radicals generated by continuous dye excitation and emission.
Materials and methods
A flow chamber was incubated with lambda phage and the viruses were allowed to settle nonspecifically on the surface. The concentration was tested by measuring the absorbance at 280 nm and the purity was verified by SDS-PAGE. The phage ejection solution (see the single-phage ejection assay above) was injected into one port, and the phages conjugated to streptavidin beads were injected into the other port at 10 L/min.
A 1064 nm laser at 100 mW was used to trap bound spheres which were moved across the boundary layer of the two solutions.
Results
A representative example of the trajectories is shown in Figure 3.3c and d — the trajectories for each ionic state are in Figure 3.6a–e. A comparison between using calibrated DNA lengths and intensity as a measure of total extracted DNA is shown in Figure 3.7a–e. As the amount of magnesium decreased and the amount of sodium increased, the extraction rate increased (figure 3.3e and f).
Additionally, we observe that the continuous and looped exhausts have velocity curves that are within error of each other (Figure 3.3e and f).
Conclusion
The theory, which assumes a crystalline packing of DNA in the capsid with repulsive forces within the strand, determined by the nature of the couterions, agrees reasonably well with the data. As previously observed [85], mobility strongly depends on the amount of DNA remaining in the capsid. Of particular importance would be models that predict the force driving emissions in different salts, in addition to a better understanding of the time scale of the emission process.
Of particular interest is the relationship of the present work to [82], which is an investigation of the packing process as a function of salts; they note that by increasing the amount of multivalent cations in their packaging assay, the virus packages faster and requires less power at an equivalent fraction of DNA packaged.
Appendix
By measuring the migration distance in each lane, we can relate the pressure exerted on the phage to the amount of DNA not remaining in the capsid, and in this way derive a pressure corresponding to the DNA that is left in the capsid as a function of amount of DNA ejected (figure 3.4a). Above each track in figure 3.9 is indicated the amount of external pressure caused by the presence of the PEG as measured in atmosphere. Migration distance in each lane was measured by searching for the maximum fluorescence peak relative to the 48.5 kbp peak, and comparing it to the corresponding ladder.
Since electrophoresis was performed in many different groups, we present the results in such a way that the relevant pressure experiment in the appropriate salt condition is to the right of the corresponding ladder.
Acknowledgements
A trend of DNA retention in the capsid with increasing external osmotic pressure is clearly visible. In the case of phage lambda, these packaging forces are sufficient to stimulate DNA ejection in vitro. In the results section we present our observations on the in vivo DNA translocation process.
We demonstrate that the time scale of DNA translocation is 1 to 20 min and that there is marked cell-to-cell variability in the dynamics.
Experimental design
By looking at this problem from a single molecule perspective, we are able to observe the cell-to-cell variability of the translocation process and gain insight into the mechanism of in vivo DNA ejection. When the parS site arrives in the cytoplasm and becomes accessible to the mCherry-ParB proteins, mCherry-ParB binds atparS, producing a fluorescent focus [142]. By complementing the dye-labeled phages with this orthogonal protocol, we independently monitor the arrival of viral DNA in the cell in the absence of fluorescent dye molecules.
This allows us to monitor the fate of the viral DNA after its arrival in the bacterial cytoplasm and watch the implementation of certain key parts of its genetic program to completion.
Materials and methods
Bacterial debris and chloroform were then removed by centrifugation for 10 min at 5000 g and recovering the supernatant. Each flask was then inoculated with 1 mL of the saturated overnight culture and placed on a 30 oC shaker until the culture reached an OD600 of ~0.6-0.8. The aqueous portion of the mixture was then recovered and passed through a 5µm filter to remove any remaining bacterial debris.
2 µL of the cell/phage mixture was then spotted onto the agar pad and allowed to dry for 5 min at room temperature.
Results
The intensity of the phage-segmented region and the cell-segmented region are each plotted separately. The red lanes show the time history of the DNA intensity in the virus, and the blue lanes show the increase in fluorescence in the cellular interior. As indicated schematically in Figure 4.1b, we simultaneously monitor phage binding to the cell through a capsid protein gpD-YFP fusion and the fate of the ejected DNA via mCherry-ParB in the host.
A binding site in the phage genome serves as a seed for localization of the mCherry-ParB fusion (figure 4.4a).
Conclusion
It is possible that a deeper understanding of the friction will advance the insights we gain from our data. As a step towards building a mechanistic picture of the ejection process in vivo in the case of phage. By simultaneously labeling the capsid proteins and the genomic DNA, it is possible to follow the viral life cycle in real time after the arrival of the viral DNA in the cytoplasm.
In particular, by monitoring the fluorescence associated with ParB proteins, we have a real-time reading of the replication process of the viral DNA.
Appendix
Dye-bound phage ejection controls
- SYTOX Orange staining of phage is equivalent to DAPI
- SYTOX-Orange–stained phage eject their DNA in vitro
- SYTOX-Orange–stained phage behave like wild-type phage in terms of bulk
- Cells in the absence of phage do not increase in fluorescence
- Photobleaching and ejection time scales are separated
- Single-phage ejection trajectories
We then show in figure 4.6b simultaneous staining and colocalization of the viral DNA with both DAPI and STYOX Orange, confirming that SYTOX Orange is both a sensitive and specific indicator for the presence of phage lambda. We observe perfect co-localization of the DAPI and SYTOX Orange signals, demonstrating that SYTOX Orange will enter the phage capsid. The presence of fluorescent puncta also provides evidence that SYTOX Orange will not adversely affect phage stability (in contrast to other dyes such as SYBR Gold) [135].
Another important question regarding the presence of the SYTOX Orange dye is the extent to which it could disrupt the dynamics of viral DNA ejection.
Strains, media, and controls for constructing λDVV1
Acknowledgments
Role of the linker between the SH2 domain and the catalytic domain in Src regulation and function. Effect of the Wiskott-Aldrich syndrome protein (WASP) C-terminus and the Arp2/3 complex on actin polymerization. Effects of salt concentration and bending energy on the extent of ejection of phage genomes.
Facilitation of bacteriophage lambda DNA injection by inner membrane proteins in the bacterial phosphoenol-pyruvate:carbohydrate phosphotransferase system (pts).
