International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 4.3, pp. 129-130   | 1 | 2 |

Section 4.3.2. Microscopic aspects of protein crystallization

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail: zsd4n@virginia.edu

4.3.2. Microscopic aspects of protein crystallization

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Proteins are inherently dynamic entities, a property that greatly hinders their crystallizability. Not surprisingly, it has been estimated that even for the stable and relatively small single-domain prokaryotic proteins fewer than one in four will yield X-ray-quality crystals when using a routine screening process (Canaves et al., 2004[link]; Price et al., 2009[link]). In order to rationally modify proteins to enhance their crystallizability, it is first necessary to understand the physical properties that make most proteins resistant to crystallization.

Protein crystals are nucleated ab initio at supersaturation levels in the 200–1000% range (McPherson, 1982[link]). Nucleation is believed to proceed via a two-step process: clusters of solute molecules form first and upon reaching critical size reorganize into three-dimensionally ordered nuclei (Georgalis et al., 1997[link]; Vekilov, 2004[link]; Erdemir et al., 2009[link]). Subsequent transfer of protein molecules from solution onto the growing crystal surface is driven by relatively small negative changes in Gibbs free energy (ΔG°), from approximately −10 to −100 kJ mol−1, at ambient temperature (Vekilov, 2003[link]). Interestingly, enthalpy changes are generally negligible during crystallization (Yau et al., 2000[link]; Petsev et al., 2001[link]; Gliko et al., 2005[link]), so that entropic phenomena dominate (Vekilov et al., 2002[link]; Vekilov, 2003[link]; Derewenda & Vekilov, 2006[link]). The microscopic effects underlying the entropy changes, both favourable and unfavourable, involve the protein itself as well as the solvent. Protein packing, which results in an ordered three-dimensional lattice and loss of translational and rotational degrees of freedom, is unfavourable and produces an energy barrier in the 30–100 kJ mol−1 range at room temperature (Finkelstein & Janin, 1989[link]; Tidor & Karplus, 1994[link]). Similarly, incorporation into the growing crystal and ordering of any intrinsically unstructured elements, such as flexible termini or loops and side chains, at the point of crystal contacts further increases the entropic cost. However, the release of ordered solvent molecules from the surfaces involved in crystal contacts, which is estimated to be in the 25–150 kJ mol−1 range, may sufficiently compensate for these entropy losses and ultimately provide the driving force for crystal growth (Vekilov et al., 2002[link]; Vekilov, 2003[link]).

Based on these considerations, it is evident that a protein must satisfy certain criteria in order to crystallize. Firstly, it must have a molecular surface that confers adequate solubility under initial conditions to reach the necessary supersaturation level for nucleation. Furthermore, it should have few, if any, intrinsically unstructured fragments such as extended N- or C-­termini or long and solvent-exposed loops which may impede crystallization. Finally, the protein should have distinct `sticky' patches on the surface with a structured layer of solvent molecules, allowing the ordering of nascent nuclei by mediating thermodynamically viable specific crystal contacts.

The notion that protein crystallization involves specific and anisotropic intermolecular interactions, as opposed to random contacts, is relatively new. Early analyses of intermolecular contacts in protein crystals concluded that crystallization is a stochastic process generated by mostly random contacts (Janin & Rodier, 1995[link]; Janin, 1997[link]; Carugo & Argos, 1997[link]). However, more recent stringent statistical analyses using a larger database strongly suggested that crystal contacts are generated by anisotropic interactions that favour small hydrophobic residues and disfavour large polar side chains with high conformational entropy (Cieślik & Derewenda, 2009[link]). This view is also supported by a large-scale comparison of the amino-acid sequences of crystallizable and noncrystallizable proteins, which established that crystallization propensity is negatively correlated with the prevalence of residues with high side-chain entropy (Price et al., 2009[link]). Finally, molecular-dynamics simulations of the intermolecular interactions of lysozyme in solution show that they are anisotropic and that their magnitude and nature depend on the physical chemistry of the participating interfaces, suggesting that the nucleation phenomenon is initiated in a nonstochastic fashion (Pellicane et al., 2008[link]).

Understanding the physical principles that govern crystallization at the microscopic level provides the singular underpinning to rationally engineer target proteins to enhance their crystallizability either by improving their solution properties or by increasing their propensity to engage in weak but specific interactions that organize the transformation of nascent clusters into nuclei and drive subsequent crystal growth.

References

Canaves, J. M., Page, R., Wilson, I. A. & Stevens, R. C. (2004). Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics. J. Mol. Biol. 344, 977–991.
Carugo, O. & Argos, P. (1997). Protein–protein crystal-packing contacts. Protein Sci. 6, 2261–2263.
Cieślik, M. & Derewenda, Z. S. (2009). The role of entropy and polarity in intermolecular contacts in protein crystals. Acta Cryst. D65, 500–509.
Derewenda, Z. S. & Vekilov, P. G. (2006). Entropy and surface eng­ineering in protein crystallization. Acta Cryst. D62, 116–124.
Erdemir, D., Lee, A. Y. & Myerson, A. S. (2009). Nucleation of crystals from solution: classical and two-step models. Acc. Chem. Res. 42, 621–629.
Finkelstein, A. V. & Janin, J. (1989). The price of lost freedom: entropy of bimolecular complex formation. Protein Eng. 3, 1–3.
Georgalis, Y., Umbach, P., Raptis, J. & Saenger, W. (1997). Lysozyme aggregation studied by light scattering. I. Influence of concentration and nature of electrolytes. Acta Cryst. D53, 691–702.
Gliko, O., Neumaier, N., Pan, W., Haase, I., Fischer, M., Bacher, A., Weinkauf, S. & Vekilov, P. G. (2005). A metastable prerequisite for the growth of lumazine synthase crystals. J. Am. Chem. Soc. 127, 3433–3438.
Janin, J. (1997). Specific versus non-specific contacts in protein crystals. Nat. Struct. Biol. 4, 973–974.
Janin, J. & Rodier, F. (1995). Protein–protein interaction at crystal contacts. Proteins, 23, 580–587.
McPherson, A. (1982). The Preparation and Analysis of Protein Crystals. New York: John Wiley & Sons.
Pellicane, G., Smith, G. & Sarkisov, L. (2008). Molecular dynamics characterization of protein crystal contacts in aqueous solutions. Phys. Rev. Lett. 101, 248102.
Petsev, D. N., Thomas, B. R., Yau, S. T., Tsekova, D., Nanev, C., Wilson, W. W. & Vekilov, P. G. (2001). Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution. J. Cryst. Growth, 232, 21–29.
Price, W. N. II et al. (2009). Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat. Biotechnol. 27, 51–57.
Tidor, B. & Karplus, M. (1994). The contribution of vibrational entropy to molecular association. The dimerization of insulin. J. Mol. Biol. 238, 405–414.
Vekilov, P. G. (2003). Solvent entropy effects in the formation of protein solid phases. Methods Enzymol. 368, 84–105.
Vekilov, P. G. (2004). Dense liquid precursor for the nucleation of ordered solid phases from solution. Cryst. Growth Des. 4, 671–685.
Vekilov, P. G., Feeling-Taylor, A., Yau, S.-T. & Petsev, D. (2002). Solvent entropy contribution to the free energy of protein crystallization. Acta Cryst. D58, 1611–1616.
Yau, S.-T., Petsev, D. N., Thomas, B. R. & Vekilov, P. G. (2000). Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals. J. Mol. Biol. 303, 667–678.








































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