Naomi E. Chayen

Protein crystallization: from purified protein to diffraction-quality crystal.

Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth.

An Automated System for Micro-Batch Protein Crystallization and Screening

An automatic sample dispenser has been constructed to aid with protein crystallization trials. This dispenser contains a bank of Hamilton syringes driven by stepper motors under computer control which is used to set up small samples (2 μl or less) for batch crystallization. Software has been written to create a series of trials which form a two-dimensional array of crystallization conditions. A specially designed fluoropolymer multibore microtip allows the very small volumes to be mixed and dispensed with great accuracy.

Microbatch crystallization under oil — a new technique allowing many small-volume crystallization trials

Abstract An approach to rapid protein crystallization using very small samples is described. A computer controlled microdispenser is used to make crystallization samples as microbatch droplets under oil. Samples of 1–2 μl are dispensed ready-mixed and with good precision. The samples are protected from evaporation, contamination and physical shock by the oil. When favourable conditions for crystallization have been found using one mode of the system, the size and quantity of crystals are optimized by a second program which generates a set of conditions throughout the area of interest. Crystals of diffraction size and quality have been grown in 1 μl drops.

The molecular basis of the coloration mechanism in lobster shell: β-Crustacyanin at 3.2-Å resolution

The binding of the carotenoid astaxanthin (AXT) in the protein multimacromolecular complex crustacyanin (CR) is responsible for the blue coloration of lobster shell. The structural basis of the bathochromic shift mechanism has long been elusive. A change in color occurs from the orange red of the unbound dilute AXT (λmax 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (λmax 632 nm in CR). Intriguingly, extracted CR becomes red on dehydration and on rehydration goes back to blue. Recently, the innovative use of softer x-rays and xenon derivatization yielded the three-dimensional structure of the A1 apoprotein subunit of CR, confirming it as a member of the lipocalin superfamily. That work provided the molecular replacement search model for a crystal form of the β-CR holo complex, that is an A1 with A3 subunit assembly including two bound AXT molecules. We have thereby determined the structure of the A3 molecule de novo. Lobster has clearly evolved an intricate structural mechanism for the coloration of its shell using AXT and a bathochromic shift. Blue/purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of β-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.

Comparative Studies of Protein Crystallization by Vapour-Diffusion and Microbatch Techniques

Numerous reports have been published in the literature which describe the crystallization of macromolecules by a variety of crystallization methods, including the vapour-diffusion and microbatch techniques. This topical review compares the results of examples of proteins which were crystallized by both vapour-diffusion and microbatch methods. The inherent features of the vapour-diffusion and microbatch methods are discussed and some specific conditions where one method appears more favourable than the other are reported. Guidelines for the conversion of crystallization conditions from vapour diffusion to microbatch (and vice versa) are also presented.

The role of oil in macromolecular crystallization

The different facets of the utilization of oil demonstrate that an individual oil and/or combinations of different oils can influence the outcome of crystallization experiments. The oil can play a part in the control of nucleation, affect the rate of equilibration and consequently determine the size of the forming crystals. Whether used for microbatch, vapour diffusion or for control of nucleation, the presence of oil is a parameter that can contribute to the accuracy, cleanliness and to the increase in the reproducibility of the experiments. Furthermore, the oil has a role in the protection of the trials during the course of their duration and in maintaining the stability of the resulting crystals.

Controlled nucleation of protein crystals

Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogeneous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase G2. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.

Crystallization with oils: a new dimension in macromolecular crystal growth

The crystal growth of biological macromolecules is a complicated process involving numerous parameters. This paper presents an approach which employs the use of oil as a major aid to crystal growth, and which has opened up a new dimension in the field of macromolecular crystallization. The presence of oil is a parameter which can contribute to the accuracy, the cleanliness and to the increase in the reproducibility of the experiments. Furthermore, the oil has a role in the protection of the trials during the course of their duration and in maintaining the stability of the resulting crystals. The use of oil also applies to the crystallization of membrane proteins. The results of a wide range of experiments which exploit the presence of oil to abet macromolecular crystal growth using both vapour diffusion and microbatch are presented.

Protein crystallization facilitated by molecularly imprinted polymers

We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as “smart materials,” are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8–10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates.

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 10 5 proteins that is underway, raises expectations for equivalent three dimensional structural databases.