Two-Step Crystal Nucleation Is Selected Because of a Lower Surface Free Energy Barrier

Abstract

Solution crystallization underpins a vast range of phenomena, from solidification of rocks in molten magma, through hardening of live tissues by the formation of biological minerals, to protein crystallization associated with a plethora of diseases. Recently, it was demonstrated that the nuclei of many important crystalline materials do not assemble directly in the solution but rather form in two steps. In the first step, disordered precursors arise, which in the second step host and enhance crystal nucleation. Here we address why two-step nucleation is preferred to direct nucleus assembly despite the low volume occupied by the precursor population. We demonstrate that nucleation of insulin crystals, which occurs with a highly regulated rate in mammalian pancreases as a part of insulin biosynthesis, is assisted by protein-rich clusters of radius 160–200 nm that occupy ca. 10–6 to 10–5 of the solution volume. We show that the surface free energy γ encountered by newly nucleated crystals is at least 10-fold lower than the independently determined surface free energy of the crystal–solution interface; the latter value dictates the barrier for direct crystal nucleation in the solution. This comparison indicates that two-step nucleation is faster than the direct pathway and is selected as a phase transformation route due to a lower surface free energy barrier and suggests that nucleation of crystals can be controlled by manipulating the properties of the disordered precursors.