Yevgeniy Kalinin

Logarithmic Sensing in Escherichia coli Bacterial Chemotaxis

We studied the response of swimming Escherichia coli (E. coli) bacteria in a comprehensive set of well-controlled chemical concentration gradients using a newly developed microfluidic device and cell tracking imaging technique. In parallel, we carried out a multi-scale theoretical modeling of bacterial chemotaxis taking into account the relevant internal signaling pathway dynamics, and predicted bacterial chemotactic responses at the cellular level. By measuring the E. coli cell density profiles across the microfluidic channel at various spatial gradients of ligand concentration grad[L] and the average ligand concentration [L] near the peak chemotactic response region, we demonstrated unambiguously in both experiments and model simulation that the mean chemotactic drift velocity of E. coli cells increased monotonically with grad [L]/[L] or approximately grad(log[L])--that is E. coli cells sense the spatial gradient of the logarithmic ligand concentration. The exact range of the log-sensing regime was determined. The agreements between the experiments and the multi-scale model simulation verify the validity of the theoretical model, and revealed that the key microscopic mechanism for logarithmic sensing in bacterial chemotaxis is the adaptation kinetics, in contrast to explanations based directly on ligand occupancy.

Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures

The dependence of radiation damage to protein crystals at cryogenic temperatures upon the X-ray absorption cross-section of the crystal has been examined. Lysozyme crystals containing varying heavy-atom concentrations were irradiated and diffraction patterns were recorded as a function of the total number of incident photons. An experimental protocol and a coefficient of sensitivity to absorbed dose, proportional to the change in relative isotropic B factor, are defined that together yield a sensitive and robust measure of damage. Radiation damage per incident photon increases linearly with the absorption coefficient of the crystal, but damage per absorbed photon is the same for all heavy-atom concentrations. Similar damage per absorbed photon is observed for crystals of three proteins with different molecular sizes and solvent contents.

Contact Line Pinning by Microfabricated Patterns: Effects of Microscale Topography

We study how the microscale topography of a solid surface affects the apparent advancing and receding angles at the contact line of a liquid drop pinned to this surface. Photolithographic methods are used to produce continuous circular polymer rings of varying cross-sectional size and shape on hydrophilic silicon wafer surfaces. Drops of water and glycerol are dispensed into the areas bounded by these rings, and critical apparent advancing and receding angles are measured and correlated with the parameters that characterize the ring cross section. For much of the examined parameter space, the apparent critical angles are independent of ring height and width and are determined primarily by the slope of the rings sidewalls, consistent with a model by Gibbs. For ring heights below a few micrometers, the critical angles decrease below the values predicted by the sidewall slopes alone. These results provide data for calculation of hysteresis on naturally rough surfaces and demonstrate a simple method for controlling and enhancing contact line pinning on solid surfaces.

A new sample mounting technique for room-temperature macromolecular crystallography

A new method for mounting protein crystals and other environmentally sensitive samples for room-temperature diffraction measurements is described. A crystal is retrieved using a microfabricated sample mount as recently reported, and the mount is inserted into a modified goniometer-compatible base. A transparent thin-wall polyester tube sealed at one end and filled with stabilizing liquid is then drawn over the crystal and sealed to the goniometer base. Compared with mounting using glass capillaries, this method can provide lower-background X-ray scattering, especially at higher resolutions; dramatically improved ease of crystal mounting with minimal chance of damage; accurate and reproducible crystal positioning relative to the goniometer base; improved crystal visibility and ease of alignment, especially for very small crystals; and compatibility with high-throughput approaches. Crystals can be rapidly screened and eliminated earlier in the data collection pipeline, and the cause of poor low-temperature diffraction can be diagnosed.

Crystal growth in X-ray-transparent plastic tubing: an alternative for high-throughput applications

A modified capillary-growth method is described that has substantial advantages for standard and high-throughput protein crystal growth. Protein-containing drops are injected into vapor-permeable flexible X-ray-transparent polyester tubing. The protein concentration in the drop increases over time by water transport through the tubing wall at a rate controlled by the wall thickness and ambient relative humidity. Unlike in conventional vapor-diffusion growth, the evaporation rate from the drop is constant over a longer time period, providing more suitable conditions for nucleation, and can be controlled by varying the tubing thickness and surrounding humidity. In situ X-ray diffraction can be performed at room temperature or, by flash-cooling, at low temperatures. Compared with glass capillaries or thick-wall plastic tubing, sealing and handling the tubing and extracting crystals are much easier.