Teresa Y. Miller

Lower dimer impurity incorporation may result in higher perfection of HEWL crystals grown in microgravity: A case study

Crystals of tetragonal hen egg white lysozyme (HEWL) grown on a series of space missions and their terrestrial counterparts were analyzed by gel electrophoresis and X-ray diffraction. The crystals were produced by vapor-diffusion and dialysis methods. The microgravity and terrestrial grown HEWL crystals were found to have effective partitioning coefficients (K eff ) for an oxidatively formed covalent dimer impurity (MW 28 K) of 2 and 9, respectively, i.e. the latter contain 4.5 times more dimers. The microgravity grown crystals allowed the collection of 24% more useful reflections and improved the resolution from 1.6 to 1.35 A. Other improvements were also noted including lower isotropic B-factors of 16.9. versus 23.8 A 2 for their terrestrial counterparts. High-resolution laser interferometry was applied quantitatively to evaluate the influence of dimer impurity on growth kinetics. It is shown that the growth of the (1 0 1) face from solution into which I % dimers were introduced decelerates with increasing solution flow rate and the growth stops at a flow rate of about 0.2 mm/s. This effect occurs faster than in ultrapure solutions. The covalently bound dimers essentially increase the amplitudes of the striation-inducing growth rate fluctuations. The effect is ascribed to the enhanced transport of growth inhibiting HEWL dimer to the interface. Theoretical analysis shows that a stagnant solution around a growing crystal is strongly depleted with respect to impurity by about 60% for the measured growth parameters as compared to the solution bulk. Thus, a crystal in microgravity grows from essentially purer solution than the ones in the presence of convection flows. Therefore, it traps less stress inducing impurity and should be more perfect. For crystal/impurity systems where K eff is small enough microgravity should have an opposite effect.

A comparison between protein crystals grown with vapor diffusion methods in microgravity and protein crystals using a gel liquid-liquid diffusion ground-based method

Abstract Crystals of human serum albumin have been successfully grown in a variety of gels using crystallization conditions otherwise equivalent to those utilized in the popular hanging-drop vapor-equilibration method. Preliminary comparisons of gel grown crystals with crystals grown by the vapor diffusion method via both ground-based and microgravity methods indicate that crystals superior in size and quality may be grown by limiting solutal convection. Preliminary X-ray diffraction statistics are presented.

PCAM: a multi-user facility-based protein crystallization apparatus for microgravity

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals.

New experimental approaches to the isoelectric fractionation of cells

Several parameters that could affect the survival rate and the resolution of cells in continuous-flow isoelectric focusing have been investigated. Cell survival at progressively lower pHs was found to be a function of the osmolarity of the medium: at pH 4 there was an absolute requirement for a 300 mOsm environment. A pH-dependent interaction of the carrier ampholytes with the cell surface has been demonstrated. The binding was very strong at pH 4, weak at pH 5 and totally abolished above pH 5. The same interaction was obtained with pentaethylenehexamine, the polyamino backbone of carrier ampholytes, stripped of carboxyl groups. A model was derived showing how at pH 4 the oligocationic carrier ampholytes, via their nitrogen groups, would bind to the polyanionic cell surface. The Hannig apparatus was adapted for cell separation in a continuously flowing curtain of isoelectric ampholytes. The medium osmolarity was maintained with glycine (delta pK 7.2), taurine (delta pK 8.5) and trimethylaminopropionyl sulphonate (TMAPS, delta pK 11). With increasing delta pK, the pH range that can be created in the presence of these compounds progressively widens, from a pH 4.3-7.5 range with glycine, to pH 3.5-7.5 with taurine up to pH 3.5-9.5 with TMAPS. The 48 fractions collected were routinely assayed for conductivity, osmolarity and pH.

Polystyrene latex separations by continuous flow electrophoresis on the Space Shuttle

Abstract The seventh mission of the Space Shuttle carried two NASA experiments in the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system. The objectives were to test the operation of continuous flow electrophoresis in a reduced gravity environment using stable particles with established electrokinetic properties and specifically to evaluate the influence of the electrical properties of the sample constituents on the resolution of the continuous flow electrophoretic device. Polystyrene latex microspheres dispersed in a solution with three times the electrical conductivity of the curtain buffer separated with a significantly larger band spread compared to the second experiment under matched conductivity conditions. It is proposed that the sample of higher electrical conductivity distorted the electric field near the sample stream so that the polystyrene latex particles migrated toward the chamber walls where electroosmosis retarded and spread the sample.