Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients.
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.
Tessier
Peter M
PM
Center for Molecular and Engineering Thermodynamics, Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA.
Johnson
Harvey R
HR
Pazhianur
Rajesh
R
Berger
Bryan W
BW
Prentice
Jessica L
JL
Bahnson
Brian J
BJ
Sandler
Stanley I
SI
Lenhoff
Abraham M
AM
D000433:1-Propanol; D000645:Ammonium Sulfate; D002845:Chromatography; D003460:Crystallization / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012965:Sodium Chloride; D012996:Solutions / Q000737:chemistry; D014461:Ultracentrifugation; D014961:X-Ray Diffraction
D004800:Enzymes, Immobilized; D012996:Solutions; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D000433:1-Propanol; D012259:Ribonuclease, Pancreatic; D000645:Ammonium Sulfate