Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium

Abstract

The determination of high-resolution structures of proteins requires crystals of suitable quality. Because of the new impetus given to structural biology by structural genomics͞proteomics, the problem of crystallizing proteins is becoming increasingly acute. There is therefore an urgent requirement for the development of new efficient methods to aid crystal growth. Nucleation is the crucial step that determines the entire crystallization process. Hence, the holy grail is to design a ''universal nucleant,'' a substrate that induces the nucleation of crystals of any protein. We report a theory for nucleation on disordered porous media and its experimental testing and validation using a mesoporous bioactive gelglass. This material induced the crystallization of the largest number of proteins ever crystallized using a single nucleant. The combination of the model and the experimental results opens up the scope for the rational design of nucleants, leading to alternative means of controlling crystallization.protein crystallization ͉ phase diagram ͉ microbatch ͉ vapor diffusion T he study of the nucleation and growth of protein crystals is one of the most important and underdeveloped areas of structural biology. Crystallization has always been, and still remains, a very difficult task, often referred to as the ''bottleneck'' of structure determination. The ability to control the nucleation phase is pivotal in the quest to overcome the bottleneck of protein crystallization (1).Protein crystals are grown from supersaturated aqueous solutions. Homogeneous nucleation takes place in the bulk of the solution, when the supersaturation is high enough for the free-energy barrier to nucleus formation to be overcome. Heterogeneous nucleation is caused by the presence in the solution of solid material, which can be an already formed crystal of the molecule to be crystallized (a ''seed'' crystal) or a different type of solid substance that has nucleation-inducing properties (a ''nucleant''). It can occur even when the supersaturation is not sufficient for homogeneous nucleation, in what is called the metastable zone of conditions (2).Because growth in the metastable zone affords kinetic advantages that often lead to larger and better-ordered crystals than those grown at higher supersaturations, an aim of protein crystallizers is to be able to induce heterogeneous nucleation (2). Thus, a search for a ''universal nucleant'' has been ongoing for 2 decades (e.g., refs. 2-4). Such a nucleant could enhance the chances of any single trial producing crystalline material.In 1988, McPherson and Shlichta (3) introduced the idea of controlling nucleation using mineral substrates as epitaxial nucleants for protein crystallization. This initiative has been pursued over the past 17 years by employing a variety of substrates (e.g., refs. 2 and 4-7), but none have proved to be generally applicable as nucleants.More recently, Chayen et al. (8) proposed the idea of using a porous substrate containing pore sizes that are comparable with the size of ...

Heterogeneous Nucleation in and out of Pores

“…Finally, only disordered porous media were effective; media in which the pores were of uniform size, such as zeolites, did not induce nucleation [9,10]. It was hypothesized [8,9] that there is a pore size at which the nucleation rate was maximal and that as disordered porous media have pores with a range of sizes a disordered porous medium is likely to have pores near this size. A zeolite's pores are all the same size and it is improbable that this size will just happen to be a size at which the nucleation is fast.…”

The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

“…1). To test the importance of immobilized precipitant on the MIPs, five model proteins, glucose isomerase (G), trypsin (T), proteinase K (P), lysozyme (L) and catalase (C), were also chosen and individual precipitants were used in accordance with their traditional crystallization trials, that is, (NH 4 ) 2 SO 4 for glucose isomerase and trypsin, Na/K tartrate for proteinase K, NaCl for lysozyme and polyethylene glycol (PEG) for catalase [15][16][17][18][19] . As shown in Supplementary Fig.…”

Carbon-Nanotube-Based Materials for Protein Crystallization

“…The ideal nucleant for protein crystallisation should have the following properties: 4 1) It should act as a nucleant for many proteins, not just one. As proteins are diverse, this is a demanding requirement.…”

“…4 In both cases the surfaces are composed of pores of typical size a few nanometres across but each pore has a different size and shape. We found that bio-glass induced crystallisation of the largest number of proteins ever crystallised using a single nucleant.…”

“…We found that bio-glass induced crystallisation of the largest number of proteins ever crystallised using a single nucleant. 4 However, the pore sizes and surface chemistry of this particular medium are not easily controlled.…”