Important progress has been made in recent years toward developing a molecular-level understanding of protein phase behavior in terms of the osmotic second virial coefficient, a thermodynamic parameter that characterizes pairwise protein interactions. Yet there has been little practical application of this knowledge to the field of protein crystallization, largely because of the difficult and time-consuming nature of traditional techniques for characterizing protein interactions. Self-interaction chromatography has recently been proposed as a highly efficient method for measuring the osmotic second virial coefficient. The utility of the technique is examined in this work by characterizing virial coefficients for ribonuclease A under 59 solution conditions using several crystallization additives, including PEG, sodium chloride, ammonium sulfate, and propanol. The virial coefficient measurements show some counterintuitive trends and shed light on the previous difficulties in crystallizing ribonuclease A. Crystallization experiments at the corresponding solution conditions were conducted by using ultracentrifugal crystallization. Using this methodology, ribonuclease A crystals were obtained under conditions for which the virial coefficients fell within the "crystallization slot." Crystallographic characterization showed that the crystals diffract to high resolution. Metastable crystals were also obtained for conditions outside, but near, the "crystallization slot," and they could also be frozen and used to collect structural information.

The osmotic second virial coefficient, B 22 , has become the quantity most widely used in developing a rational understanding of protein crystallization. In this work a novel method of measuring B 22 using self-interaction chromatography (SIC) is presented that is at least an order of magnitude more efficient than traditional characterization methods, such as static light scattering. It is shown that SIC measurements of second virial coefficients for BSA are in quantitative agreement with static light scattering results. The measured virial coefficient for both BSA and myoglobin reveal a surprisingly narrow range of concentrations of ammonium sulfate that promote weakly attractive interactions that are optimal for crystallization. Using the virial coefficient information, myoglobin crystals were obtained by ultracentrifugal crystallization in a rational and rapid manner.

The pore structure of chromatographic adsorbents directly influences macromolecular partitioning and transport in chromatography. Quantitative structural characterization of chromatographic media has generally been performed in terms of the mean pore size or, at best, the pore size distribution (PSD), but more detailed information on, e.g., connectivity has been lacking. We have applied electron tomography, a 3D TEM technique that views a sample from multiple perspectives and allows reconstruction of the volumetric structure, to capture the internal details of microporous chromatographic media with nanometer-scale resolution. Visualization of reconstructions of three adsorbents, Toyopearl SP-650 C, SP-550 C, and CM Sepharose FF, provides thorough and direct information on the geometry and the interconnectivity of the pore network. The structures are qualitatively consistent with in situ AFM images, and quantitative data for the porosities and PSDs from the analysis of tomographic data agree reasonably well with inverse size-exclusion chromatography results. For a more straightforward representation of the networking and size features of the disordered pore space, a 3D thinning algorithm was used to derive pore skeletons and consequently quantitative data on distributions of local path lengths, widths, tortuosities, and connectivities. Such enriched structural information can be instrumental in more discriminate structural evaluation and construction of engineered pore models for the study of solute intraparticle transport.