It has long been known that certain solid additives, i.e., a heterogeneous nucleant (HN), can facilitate the deposition of crystals from solutions or melts. Often times, in spite of the fact that the solution to be crystallized is supersaturated with respect to solute, an initial nucleation event does not occur and therefore there is no subsequent crystal growth. An efficacious heterogeneous nucleant can assist, by decreasing the translational motion of species in solution by adsorbing them onto its surface, in overcoming the entropic barrier which allows ordering of the solution species into a 3-D crystal. Examples include the seeding of solutions with the crystals to be formed, chemists scratching glass flasks to induce crystallization, the rapid freezing of supercooled water by the addition of ice and the formation of rock candy on strings dipped into concentrated sugar solutions. More recently, “debris-based nucleants” such as ground glass, seaweed, hair, polymer spheres and other detritus, which could exert unknown deleterious effects on the crystallizing proteins, have been used in attempts to increase crystallization efficacy. Since the structural information obtained by determining the 3-D X-ray crystal structures of crystallized proteins is invaluable in determining protein function, and there is generally a very low chance of obtaining X-ray diffraction quality single crystals in any given crystallization trial, there is an urgent and immediate need for materials to initiate and facilitate the crystallization of proteins. One way to encourage, control, facilitate, accelerate, and provide selectivity in terms of polymorph crystallized, is to use a heterogeneous nucleant.
In order for any crystal to form from solution there must be an initial phase where there is only sub-nanometer sized solid assemblages (nuclei) of the soluble species (e.g., protein molecules) aggregating into a lattice in an ordered 3-D arrangement in space called a crystal. Most often, the reason that a given soluble species cannot crystalize, when the solution is already saturated or supersaturated, is that there is no efficient nucleation mechanism available to induce the initial order required to commence crystal growth. In principle, this nucleation event may occur within the solution itself (homogeneous nucleation) or on the surface of the solution container or on an adventitious particle within the solution (heterogeneous nucleation). The homogeneous nucleation depends on factors such as temperature, concentration, pH or solution composition whereas the heterogeneous nucleation depends on these parameters but also on the nature of the surface on which the initial nucleation events takes place. In order to increase the chances of obtaining useful crystals beyond homogeneous nucleation it would be necessary to use heterogeneous nucleation since the homogeneous nucleation is already a subset of every heterogeneous nucleation experiments.
There is no universally accepted explanation for the mechanism by which a heterogeneous nucleation catalyst performs its function of facilitating protein crystallization. It is clear, since the crystal is comprised of a regular spatial array of molecules or substituents, and these incipient crystal components are well separated in the solution or melt, that the constituents must lose translation degrees of freedom and some amount of entropy in order to form the solid. So in some sense, the heterogeneous nucleation catalysts likely provide a surface onto which the soluble constituents may attach themselves thereby reducing their translation freedom. Once absorbed a certain amount of surface mobility is required of the protein. The heterogeneous nucleant must allow enough surface diffusion of the proteins molecules for them to “anneal” into their preferred orientation in the lattice. If the adsorption is too weak and fleeting to induce order, or so strong that the protein is adsorbed in an irreversible manner in a random orientation, then there is no crystalline nucleus onto which the protein molecules may adsorb to grow the crystal.
Fig. 1 A crack on a substrate surface begins with a finite width which tapers to zero width as the crack terminates within the substrate.
A heterogeneous nucleant (HN) can influence crystal growth in many ways but the fundamental effect must be to form a single type of nucleus at the earliest stages of nucleation and crystal growth. It seems clear that once a given type of nucleus forms, other crystalline polymorphs or nuclei of differing habit cannot form and are all other nuclei polymorphs but one are completely suppressed. There are many examples in the Image Gallery showing the strong influence of the Photon Hammer nucleants on crystal growth in the ways enumerated below. In all of the examples shown, which provides a summary of hundreds of heterogeneous nucleation experiments, there was never an example of two different crystal habits, types or polymorphs observed within the same crystallization solution. This is consistent with only one polymorph existing in the solution in the presence of the heterogeneous nucleant.
A heterogeneous nucleant is generally a solid state material which, when in contact with a solution containing the soluble species to be crystallized, alters the course of crystallization as compared to the course in the absence of the HN in ways such as:
Fig. 2 Myriad plumate microcracks are created using Parallel’s unique Photon Hammer heterogeneous nucleation technology. Note that each crack has a finite width as it emanates from the laser damage zone but tapers to zero width, in directions both parallel an perpendicular to the substrate surface, as it terminates a short distance away. Presumably, at some point along the crack, there exists a space of the correct size that is capable of adsorbing protein molecules and reducing their translational motion. The reduced translation motion could assist in forming the nascent nuclei required for further crystal growth.
We hypothesize that the cracks resulting from the Photon Hammer fabrication process (Fig. 2) provide a geometrical environment suitable for ordering the soluble protein molecules into incipient nanoscopic nuclei.
Cracks with a tapering morphology, where the cracks taper from a finite width at the site of crack initiation to zero width at the termini of each crack, could provide a geometry favorable for the ordering of the protein molecules into a 2-D raft-like structure capable of nucleating further crystal growth.
Examples of most of the above-described heterogeneous nucleation phenomena may be found within the Image Gallery section of this website.