Direct determination of the nucleation rates of protein crystals

We have developed a novel method for direct determination of the steady-sta te rates of homogeneous nucleation. The method is applicable to studies of crystallization, aggregation, and similar first-order phase transitions in solutions of proteins or other soluble slow-growing materials with temperat ure-dependent solubility. Temperature T control of the solution supersatura tion allows fast supersaturation changes from a level inductive of nucleati on to a level where no nucleation occurs, but existing crystals grow to det ectable dimensions. In this way, nucleation takes place only at the first T setting at a constant supersaturation before solution depletion due to cry stal growth becomes significant. We use inert oil to cover nucleating solut ions and suppress nucleation on the solution-air interface. To obtain repro ducible statistical characteristics of the intrinsically random nucleation process, a large number of simultaneous trials take place under identical c onditions. First data for the nucleation of the model protein lysozyme unde r typical crystallization conditions show that protein crystal nucleation m ay be occurring at or even beyond the boundary of applicability of classica l, continuum nucleation models.