The crystallization of biological macromolecules from precipitates: Evidence for Ostwald ripening

Crystals were obtained by different methods under conditions where nucleation and growth occur from precipitated macromolecular material. The phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an alpha-amylase, a thermostable aspartyl-tRNA synthetase, the nucleo-protein complex between a tRNA(Asp) transcript and its cognate yeast aspartyl-tRNA synthetase, and tomato bushy stunt virus, In each system, after a rather rapid precipitation step at high supersaturation lasting one to several days, a few microcrystals appear after prolonged equilibration at constant temperature. With alpha-amylase, the virus and the thermostable synthetase, crystallization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by Ostwald ripening. In the case of thaumatin, concanavalin A and the nucleo-protein complex, crystallization occurs by a phase transition mechanism since it is never accompanied by the disappearance of the smallest crystals. A careful analysis with thermostable aspartyl-tRNA synthetase indicates that its crystallization at 4 degrees C under high supersaturation starts by a phase transition mechanism with the formation of small crystals within an amorphous protein precipitate. Ostwald ripening follows over a period of up to three/four months with a growth rate of about 0.8 Angstrom/s that is 13 times slower than that of crystals growing at 20 degrees C in the absence of precipitate without ripening. At the end of the ripening process at 4 degrees C, only one unique synthetase crystal remains per microassay with dimensions as large as 1 mm.