Researchers team have revealed for the first time uncovered the molecular details of protein crystal nucleation, a process with great medical and scientific relevance. The team also developed a new methodology to study a broad class of systems that have remained elusive to date. The study was published in Nature.

Medical importance of protein crystals

Protein crystals bear great medical and scientific relevance. For decades, they have been essential for structural biologists to solve the three-dimensional structures of proteins, but protein crystals are also used as bio-pharmaceutical delivery agents.

Crystalline suspensions are attractive formulations to store and administer active pharmaceutical compounds because of their long-term shelf life, low solvent viscosity, and slow dissolution rate. Perhaps the best-known example is insulin.

Insulin shots comprise the subcutaneous injection of a suspension of insulin microcrystals which dissolve slowly to yield a steady and sustained delivery over time. Despite their tremendous potential, there are two factors that limit the use of protein crystals in a broad range of applications.

Challenges in developing protein crystals

First, growing protein crystals, as many molecular biologists will say, is more an art than a science. In fact, for many proteins, crystallization can be excruciatingly difficult. This in part follows from the fact that scientists don't understand the early stages of protein crystal formation.

Any crystal originates from a nucleus, a tiny crystalline seed, which forms by the spontaneous grouping of a few molecules in solution that must adopt a regular organization in three-dimensions. How the molecules realize this improbable feat has remained a mystery up until this point.

Secondly, a single protein can crystallize in multiple different crystal forms, this is known as polymorphism. Different crystal polymorphs have different characteristics, with the most notable ones the power to diffract X-rays (crucial for 3D structure determination), and the rate at which it dissolves.

A new way to look at the self-assembly of macromolecules

The group of scientists has used state-of-the-art cryo-transmission electron microscopy (Cryo-TEM) to capture the birth of a protein crystal by visualizing the process of nucleation at molecular resolution. By analyzing the Cryo-TEM images taken from a series of samples at constant time intervals.

They could start to puzzle together the series of molecular collisions that need to take place to form a crystalline nucleus. By analyzing and understanding the differences in the structure of the various nuclei, they developed strategies to guide the polymorph selection process.

They achieved this by gently tweaking the different modes of interaction that exist between the molecules, steering the nucleation process in the direction of our choosing. The team believes that the new insights and methodology will significantly advance the development of protein crystals for 3D structure determination and medical applications.