Protein crystallization

3 3 M M E E T T H H O O D D S S

3.1 Protein crystallization

For proteins larger than 30 kDa the only possibility to get atomically resolved structural information is to obtain single crystals and subsequent collection of X-ray diffraction data. The main obstacle in this procedure is the identification of conditions under that the protein of interest will crystallize. A comprehensive overview of crystallization techniques is given in literature (Bergfors, 1999). A typical crystallization experiment is set up by mixing a “high”

protein concentration with several precipitants to be tested. These precipitants are quite diverse in nature and can be: salts (e.g. NaCl; (NH4)2SO4), organic compounds (e.g. polyethylene glycols, methyl pentanediol), organic solvents or various combinations thereof. Initially the precipitant concentration is chosen to be lower than would be necessary to precipitate the protein. This point has to be found for every protein/precipitant-combination empirical. Subsequent, the condition is allowed to slowly increase the concentration of protein and precipitant by controlled evaporation of water. Although several approaches exist to achieve this, the most common used one is vapor diffusion. Here the miniaturized condition with protein and precipitant is enclosed together with a larger reservoir of undiluted precipitant (either as

“hanging” or “sitting drop”). The sealing guarantees that water will slowly evaporate from the drop, as long as the precipitant concentration in the drop is lower than in the reservoir. Thus, the drop shrinks and both, the precipitant and the protein concentration are increased. Figure 7 shows and describes an idealized phase diagram for a vapor diffusion experiment. Note that for each examined precipitant, concentrations have to be optimized in order to prevent precipitation and avoid undersaturation. Another typical problem is associated with the fact that nucleation and (optimal) crystal growth are not typically occurring at the same position of the phase diagram. Hence, a too steep increase of concentrations might lead to overnucleation and formation of many small and bad diffracting crystals. Although not usable for the diffraction experiment itself, these tiny crystals still might pave the way to success by serving as starting material in various kinds of seeding experiments. By preventing the need to bring the system to a state where nucleation occurs, these seeding experiments can typically speed up the crystallization experiment and could yield bigger crystals than obtainable without seeds.

Up to date identification of a precipitant condition that gives an initial hit that could be optimized further is still an empirical approach. Nevertheless, automation and miniaturization allows the fast screening of various conditions without too much effort. Typically, screens are available that comprise various conditions (i.e. combinations of different precipitants, additives and buffers), which have been used successfully in crystallization trials of other proteins. These biased, random screens are called sparse matrix screens. Additionally, screens are available that try to rationalize the sampling of the parameter matrix (Grid screens). Caused by the huge

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amount of parameters (e.g. precipitants, additives and buffers with various concentrations and in different combinations), an entirely systematic screen is not feasible and Grid screens deploy their strength mainly in the optimization of an identified initial hit.

Figure 7: Phase diagram of an optimal protein-precipitant condition for vapor diffusion. The axes represent the concentration of precipitant and protein, respectively. The vapor diffusion experiment starts at point 1, where concentrations of protein and precipitant are low enough to ensure protein’s solubility. Due to evaporation of water, the drop’s volume shrinks and the concentration of protein and precipitant are equally increased. As soon as the nucleation zone is hit (2), crystal nuclei will form and thus deplete the protein concentration, which brings the condition to a phase of slow and even crystal growth (3).

Despite the technical improvements made to enable automatized high-throughput screening of various conditions, new statistics show that only 10% of all proteins will crystallize readily (Kim et al., 2008) and that those will most likely show a first promising result even if screening only a small set of different conditions (Z.S. Derewenda, 2004). Thus it would be hardly meaningful to uninspiredly extent the conditions screened, in order to find a suitable condition for a protein that resisted successful crystallization so far. Besides this random trial and error approach with excessive testing of various possible crystallization conditions, one can think of various improvements of the protein in order to yield well diffracting crystals. These include:

•reductive methylation of surface exposed lysines (Kim et al., 2008)

•surface entropy reduction (Z.S. Derewenda and Vekilov, 2006; Cooper et al., 2007)

•construction of fusion proteins/ fixed arm carrier (Smyth et al., 2003; Moon et al., 2010)

•proteolytic digestion/ removal of flexible parts (Wernimont and Edwards, 2009)

•cocrystallization with ligands

•crystallization of homologue (thermostable) proteins

•antibody mediated crystallization (Hunte and Michel, 2002)

•GraFix/ cross linking approach to yield monodisperse complexes for 3D-cryo EM (Kastner et al., 2008)

•a combination of the aforementioned approaches (Moon et al., 2010)

[precipitant]

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In the following the advantages and disadvantages of the single techniques will be discussed. In order to form a well ordered crystal lattice the sample has to be homogenous and should consist of molecules with a defined surface that can interact with each other. The first two approaches to optimize the protein, utilized for crystallization, follow the same idea: Charged side chains with long, and therefore intrinsically not well ordered, sidearms are modified in order to facilitate regular contacts between individual protein molecules. Especially for peripheral membrane binding proteins this seems to be of high importance, because these proteins have large, positively charged clusters on their surface, which are supposed to interact with the anionic phospholipids of the membrane (Bhardwaj et al., 2006). While the first approach aims at a biochemical methylation and will only affect surface exposed residues, the second approach consists of site-directed-mutagenesis steps to replace surface exposed glutamic acids and lysines with alanine. This implies all the drawbacks, one always have to keep in mind when dealing with site-directed-mutagenesis. Moreover the identification of surface exposed residues without structure is somewhat empirical and a large surface area might require the mutation of different amino acids, with each and every mutation step having the same inherent threat of structure perturbation or loss of function. While these two approaches modify the protein surface to allow the formation of crystal contacts, the other approaches rather try to yield monodisperse and well structured units for the formation of the crystal lattice. Therefore unordered loops and tails can be removed by limited proteolysis of the native protein. Upon substrate binding enzymes might undergo an induced fit, yielding a higher ordered structure. Another technique that proved to be very valuable for 3D cryo EM is called GraFix. The idea is to obtain monodisperse particles by ultracentrifugation and simultaneous fixation of this state by cross linking with glutaraldehyde. The promise of employing fusion proteins is that the added protein domain will crystallize readily and provide crystal surfaces that might guide the crystallization of the enzyme of interest. Furthermore the structure of the fused protein often is resolved to high resolution and this information can be used to ease the problem of phase determination. Unfortunately, the generated multidomain proteins are often very flexible with respect to the arrangement of the domains. Therefore a rigid linker is needed to assure a homogenous domain architecture, which in turn is a prerequisite for the formation of a defined crystal lattice (Smyth et al., 2003).

Fragments of antibodies, raised specifically against the protein of interest can fixate intrinsically disordered, flexible parts of the protein, shield hydrophobic regions and provide new surfaces for crystals. Although nowadays generation of antibodies has become a standard technique, it is still expensive, labor-intensive and time-consuming. If modification of the protein does not lead to well diffracting crystals, a last opportunity is to crystallize a homologue protein. This related protein might be less flexible and therefore more easily to crystallize.

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