Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients

Abstract

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.

Colloidal Behavior of Proteins: Effects of the Second Virial Coefficient on Solubility, Crystallization and Aggregation of Proteins in Aqueous Solution

“…Compared to SLS, SIC requires at least an order of magnitude less time and sample [22], permits the use of high molecular weight or selfassociating co-solvents [26], allows virial coefficient measurements to be made on small biomolecules (i.e. peptides), and has demonstrated the potential for miniaturization [27].…”

Protein aggregation in salt solutions

“…The second virial coefficient is a principal measure of pairwise proteinprotein interactions in solution. In recent years, the second virial coefficient has become an important tool for understanding and predicting protein crystallization conditions (28,29,(44)(45)(46)(47)(48)(49)(50)(51)(52). George and Wilson (44) were the first to notice that the conditions that best promote protein crystallization are those that fall within a particular "crystallization slot" of values of the second virial coefficient, B 22 Rarely are experimental measurements made on a given type of protein of all of the properties of aggregation together-the cloud point, the liquid-liquid phase coexistence curves, the second virial coefficient, and/or the osmotic compressibility.…”

New monoclinic form of bovine pancreatic ribonuclease A from a salt solution and comparison of intermolecular interactions in ribonucleases A

“…The same attempt was carried out using self- (9) or M3-M4 (10) ), light blue (M2-M1 (7) or M4-M3 (8) ) and bluish-white (M2-M2 (7) , M2-M2 (9) , M4-M4 (8) or M4-M4 (10) ); interaction II in the M form, dark green (M1-M2 (5) or M3-M4 (6) ), light green (M2-M1 (3) or M4-M3 (4) ), and yellow (M1-M1 (3) , M1-M1 (5) , M3-M3 (4) , or M3-M3 (6) ); interaction III, purple; interaction IV, gray. Superscripts in this legend refer to the symmetry operations given in the footnote of Table 2. interaction chromatography, where the protein-protein interactions of RNase A under various solution conditions were measured in terms of the osmotic second virial coefficients [30]. Attractive protein-protein interactions were revealed to increase with the salt concentration.…”