Engineering topics

Thomas, Bill R.

Publications and source records attributed to Thomas, Bill R..

3 records

NASA NTRS2000

Direct Visualization of an Impurity Depletion Zone

When a crystal incorporates more impurity per unit of its volume than the impurity concentration in solution, the solution in vicinity of the growing crystal is depleted with respect to the impurity I,2. With a stagnant solution, e. g. in microgravity or gels, an impurity depletion zone expands as the crystal grows and results in greater purity in most of the outer portion of the crystal than in the core. Crystallization in gel provides an opportunity to mimic microgravity conditions and visualize the impurity depletion zone. Colorless, transparent apoferritin (M congruent to 450 KDa) crystals were grown in the presence of red holoferritin dimer as a microheterogeneous impurity (M congruent to 900 KDa) within agarose gel by counterdiffusion with Cd(2+) precipitant. Preferential trapping of dimers, (distribution coefficient K = 4 (exp 1,2)) results in weaker red color around the crystals grown in the left tube in the figure as compared to the control middle tube without crystals. The left and the middle tubes contain colored ferritin dimers, the right tube contains colored trimers. The meniscus in the left tube separate gel (below) and liquid solution containing Cd(2+) (above). Similar solutions, though without precipitants, were present on top of the middle and right tube allowing diffusion of dimers and trimers. The area of weaker color intensity around crystals directly demonstrates overlapped impurity depletion zones.

Chernov, Alex A.↗

NASA NTRS1998

Effects of Convective Solute and Impurity Transport in Protein Crystal Growth

High-resolution optical interferometry was used to investigate the effects of forced solution convection on the crystal growth kinetics of the model protein lysozyme. Most experiments were conducted with 99.99% pure protein solutions. To study impurity effects, approx. 1% of lysozyme dimer (covalently bound) was added in some cases. We show that the unsteady kinetics, corresponding to bunching of growth steps, can be characterized by the Fourier components of time traces of the growth rate. Specific Fourier spectra are uniquely determined by the solution conditions (composition, temperature, and flow rate) and the growth layer source activity. We found that the average step velocity and growth rate increase by approx. I0% with increasing flow rate, as a result of the enhanced solute supply to the interface. More importantly, faster convective transport results in lower fluctuation amplitudes. This observation supports our rationale for system-dependent effects of transport on the structural perfection of protein crystals. We also found that solution flow rates greater than 500 microns/s result in stronger fluctuations while the average growth rate is decreased. This can lead to growth cessation at low supersaturations. With the intentionally contaminated solutions, these undesirable phenomena occurred at about half the flow rates required in pure solutions. Thus, we conclude that they are due to enhanced convective supply of impurities that are incorporated into the crystal during growth. Furthermore, we found that the impurity effects are reduced at higher crystal growth rates. Since the exposure time of terraces is inversely proportional to the growth rate, this observation suggests that the increased kinetics instability results from impurity adsorption on the interface. Finally, we provide evidence relating earlier observations of "slow protein crystal growth kinetics" to step bunch formation in response to nonsteady step generation.

Vekilov, Peter G.↗

NASA NTRS1996

Repartitioning of NaCl and Protein Impurities in Lysozyme Crystallization

Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth- repartitioning model, suggests a core of approx. 40 microns in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 pm in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

Vekilov, Peter G.↗