The nucleation of protein crystals as a race against time with on- and off-pathways

High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydro­dynamic radius of ∼100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals.