Krista Freeman

Size and Shape Characterization of Thermoreversible Micelles of Three-Armed Star Elastin-Like Polypeptides

Three-armed star elastin-like polypeptides are shown to have the capability of self-assembling into micellar constructs at certain environmental conditions. Here, a study of the size distribution, shape, and molecular weight of these micelles at different salt concentrations and pH values is presented. Multiangle dynamic light scattering was used to study the formation, reversibility, and size of the micelles at different environmental conditions. On the basis of the salt concentration of the solution, two distinct size distribution regimes and a transition region were observed. Static light scattering was performed to study the molecular weight and geometrical anisotropy of the micelles in each regime. The anisotropic behavior and elongation of the particles were independently confirmed by depolarized dynamic light scattering, and a model for micelles at each regime was proposed. The size and molecular weight of the micelles were verified using viscosity measurements. The results of this study suggest that there is big jump in the size and molecular weight of the micelles from the first salt-dependent regime to the other, and the shape of the micelles changes from spheres to cylindrical micelles with a higher than 10:1 axis ratio.

Portal Stability Controls Dynamics of DNA Ejection from Phage

Through a unique combination of time-resolved single-molecule (cryo-TEM) and bulk measurements (light scattering and small-angle X-ray scattering), we provide a detailed study of the dynamics of stochastic DNA ejection events from phage λ. We reveal that both binding with the specific phage receptor, LamB, and thermo-mechanical destabilization of the portal vertex on the capsid are required for initiation of ejection of the pressurized λ-DNA from the phage. Specifically, we found that a measurable activation energy barrier for initiation of DNA ejection with LamB present, Ea = (1.2 ± 0.1) × 10(-19) J/phage (corresponding to ∼28 kTbody/phage at Tbody = 37 °C), results in 15 times increased rate of ejection event dynamics when the temperature is raised from 15 to 45 °C (7.5 min versus 30 s average lag time for initiation of ejection). This suggests that phages have a double fail-safe mechanism for ejection-in addition to receptor binding, phage must also overcome (through thermal energy and internal DNA pressure) an energy barrier for DNA ejection. This energy barrier ensures that viral genome ejection into cells occurs with high efficiency only when the temperature conditions are favorable for genome replication. At lower suboptimal temperatures, the infectious phage titer is preserved over much longer times, since DNA ejection dynamics is strongly inhibited even in the presence of solubilized receptor or susceptible cells. This work also establishes a light scattering based approach to investigate the influence of external solution conditions, mimicking those of the bacterial cytoplasm, on the stability of the viral capsid portal, which is directly linked to dynamics of virion deactivation.