Perspectives for Pure and Applied Protein Crystallogenesis Studies Bernard Lorber* De´ partement ‘Me´ canismes et Macromole´ cules de la Synthe` se Prote´ ique et Cristallogene` se’, UPR 9002 du CNRS, Institut de Biologie Mole´ culaire et Cellulaire, 15, rue Rene´ Descartes, 67000 Strasbourg, France
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 1 17-19
Received April 26, 2004
ABSTRACT: The unpredictability of protein crystallization is essentially due to our ignorance of the correlations existing between architectural features of these macromolecules and physical chemical properties of the crystallization medium in which their crystals nucleate and grow. Some ideas are suggested to guide crystal growth and bioinformatics specialists in their quest of relationships that might help to rationalize the general approach of protein crystal growth. The last 50 years have seen spectacular advances in various fields of scientific research. Planetary exploration has provided us with sharp pictures of remote celestial bodies, and archeological discoveries of intermediate ancestors have helped us to trace back our own speciation. The things are apparently different when one looks at protein crystallization. In particular, the manner of how the ab initio crystallization of a novel protein is faced nowadays remains rather empirical.1-4 As in earlier times, crystal growers feel their way by trial and error when they search for the appropriate chemical composition of a solvent that will hopefully trigger crystal nucleation and growth. The above criticism may seem severe and exaggerated to experienced experimenters because of the inordinate difficulty encountered daily when performing this task. Anyhow, it is astonishing how little we know about the pathway followed by billions of molecules when they interact within a test tube and stack in three dimensions to form the solid edifices we call crystals. In fact, the situation well illustrates that all beforehand structural knowledge, available through primary structure analysis (like amino acid sequence and composition, net charge, isoelectric point, hydrophilicity, etc.) or through secondary and tertiary structure prediction algorithms, is finally insufficiently correlated to crystallizability data. As a consequence, we hardly know why some proteins never produce crystals or why others are not easily reproducible. Further, we can only guess why irregularly shaped crystals sometimes unexpectedly (some dare say “by enchantment” or even “miraculously”) have much better diffraction properties than regularly faceted ones. Traces of impurities are generally admitted to cause defects alike in crystals made of small molecules.5 Ultrasensitive biochemical purity and homogeneity analyzes may be required to assert the presence of such contaminants. In addition, crystal quality frequently decays once growth has ceased. Beyond this, it is unclear what distinguishes macromolecules producing crystals diffracting X-rays at a subatomic resolution from others and what makes that the latter crystals are fundamentally different. * Phone: +333 8841 7008.Fax: +333 8860 2218.E-mail: B.Lorber@ ibmc.ustrasbg.fr.
Two decades of extensive and careful light- and X-ray scattering investigations were necessary to prove that pure and monodisperse protein solutions have a higher propensity to crystallize.6,7 Afterward, it was observed that there is a “crystallization slot” within which enhanced attractive interactions in such solutions give birth to crystal nuclei once appropriate conditions are met.8,9 At the moment, no other test is available that can serve as a guide and speed up the search of the “best” solvent. As to the scoring of the crystallization outcome, it is not at all standardized, and experimenterto-experimenter variation in the evaluation may be misleading, especially when a quality label has to be assigned to precipitates. A great number of data on crystal growth rates and mechanisms have been gained using various microscopy methods. More investigations are desirable on a broader panel of molecules to convince a wider audience about the usefulness of crystal quality optimization methods (such as growth in gels or under microgravity). Finally, theories, be they controversial or not, should absolutely be supported by more wet-lab testing. The challenge of protein crystallization has gradually glided from crystal preparation itself to protein solubilization because these pure biological materials frequently tend to form aggregates that hamper the growth of well-ordered crystals. The solubilization step is the real bottleneck of many current structural projects. Again, it shows our ignorance of the n-dimensional space in which these macromolecules are folded in their native conformation, are stable, and retain fully their original biological function. Aggregation seems to be a less frequent problem with nucleic acids, but pronounced flexibility and partial melting are often other obstacles to overcome. Solubility is a function of macromolecular structure and of solvent components.10 Besides the negative experience gained during the handling of novel molecules, usually almost nothing is known about the limits of their stability. Systematic investigations to map the elliptic stability zone in 2D diagram representing, e.g., pressure v. temperature, or temperature v. ionic strength or ionic concentration11 is certainly not more time-consuming than doing assays at random. With regard to macromolecules found in
10.1021/cg030065o CCC: $30.25 © 2005 American Chemical Society Published on Web 09/14/2004
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extremophiles that exhibit a remarkable stability under extreme temperatures, pressures, salinities, or pHs, conventional ones that are adapted to more normal conditions may reserve surprises. Some have hidden characteristics that can compete with those of the former ones. Information Wanted for More Successful Crystallizations. Nowadays, the sparse matrices12,13 that were designed in the 80s to circumvent the impossibility to prepare manually quasi infinite numbers of assays seem to be old fashioned. Although screens encompass ever increasing numbers of formulations to augment the numbers of parameters and of levels, their actual limit is the variety of the chemical ingredients composing them. Lately, automation and miniaturization make it possible to set up within hours myriads (etymologically “tens of thousands”) of crystallization assays in microor even nanovolumes.14 Obviously, this increases the chance of finding rapidly at least one that produces exploitable crystals, if the chosen range of variables is broad enough. Thereby, the quantity of biological material scaled down by 2 orders of magnitude is less often a hindrance. On the other hand, studies comparing the effects of physical or chemical variables on more than one small and monomeric model protein are scarce until now.15 Likewise, published phase diagrams of protein concentration v. precipitant concentration displaying the distribution of polymorphs are rare.17 Fortunately, giant databases accumulate every day the positive and the negative results of millions of crystallization trials. Proteins designed by nature to fold and function properly in prokaryotic cells differ to some extent from their eukaryotic counterparts and vice versa. Similarly, the architecture of proteins from subcellular organelles evolved in environments that subjected them to constraints differing from those in the cytoplasm. Therefore, proteins belonging to a same family or accomplishing a similar function inside a given cellular compartment could share similarities at the level of their surface properties and thus of their solubility. Such trends might be revealed by plotting the statistical distribution of successful protein crystallizations against a given physical chemical parameters. To shed light on the intricacy of the effects of precipitants, it is thus desirable to examine a wider range of experimental variables than just the chemical nature of the ones yielding the highest scores.15 There is a chance to recognize subtler effects. For instance, correlations might exist with other ingredients of the crystallization medium, such as ions, buffering substances, or peculiar properties such as the chain length of polymeric precipitants, etc. Hofmeister’s demonstration that salt ions can be ranked in series with regard to their efficiency in precipitating egg-white proteins (i.e., half a century before the first pure protein was crystallized) was followed by thorough analyses whose results have strengthened our view.16 Nonetheless, no general classification of crystallization hits has been reported, neither against any property of individual ions (e.g., radius or strength) nor against any rheological property (e.g., viscosity, surface tension, or osmotic pressure) or characteristic (e.g., surface tension, dielectric constant) of the solvent. Proteins are macroions, and the comparison of the behaviors of variants, isozymes, or mutants might be a straightforward means
Perspective
to elucidate the role of a solvent through a systematic variation of its major and accessory components, to superpose packing contacts, obtain thermodynamics data, and propose crystal growth models. Moreover, it is of paramount interest to find out (i) if there is any preferential temperature (independently of the one of crystal growers) as in the case of short nucleic acids that readily crystallize at high temperatures, i.e., 37 °C, and (ii) how crystallizability varies with the pH (since solubility is minimal at the isoelectric point). In the supercomputer age, it should not take too much time to crunch experimental data coming from crystallizations and from structure analyses to look for close relationships. An excellent data sample to start with is the one contained in the Protein Data Bank18 (and in the related Biological Macromolecule Crystallization Database19). Its 27,000 structures and the corresponding successful crystallization conditions are as many potential Rosetta stones waiting on future Champollions trying to decipher the rules of protein folding and of crystallogenesis. High throughput crystallizations performed at industrial scale in the frame of structural genomics projects continuously add huge numbers of fresh data. As for inorganics, a few preferential space groups have been identified for proteins.20 It is unclear if this selection is precipitant dependent. To comprehend the relative importance of each physical chemical variable, it is necessary to quantitate separately for all crystallization conditions the solvent properties that are presently missing in databases. In a second step, the combination of structural information (e.g., nature and degree of exposure of the chemical groups of surface residues or of residues involved in packing contacts) with above physical chemical information will surely give fascinating results about the molecular characteristics that determine crystallizability. A particular attention should be paid to crystals with an exceptional degree of perfection and to those with unusual volume suitable for neutron diffraction studies. To conclude, it is the right time for a transition from the state of an art to that of a science to occur in protein crystallization. All resources are in principle available. There is quite a lot to do to explore the contribution of each variable of the multiparametric process, and it is sure that a considerable effort and much energy will be needed to unravel the remaining mysteries. In this quest of a universal remedy, the possibility to effectively orient the search of crystallization conditions for proteins possessing common surface properties is a first reward. It will represent only a step toward the ultimate goal that is to know in advance, to master, and to control the crystal growth of any target macromolecule or multimacromolecular complex. This driving force should be sufficient to sustain several bioinformatics projects. It can be anticipated that the number of crystallographers will be limiting once the avalanche of new 3D structures to solve for biology and medicine will be triggered. Acknowledgment. I dedicate this paper to the late physicist and virologist Jean Witz for unforgettable discussions about the history of protein and virus crystallization.
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