Crystallization of Biomolecules

Introduction

The elucidation of a macromolecular structure to the atomic level by X-ray or neutron diffraction analysis requires the molecule to be available in the form of relatively large single crystals. Many soluble proteins, membrane proteins, nucleic acids and nucleoprotein complexes have been obtained in a crystalline form suitable for crystallographic investigation.^1-12

If a solution of a biopolymer is brought to supersaturation, the biopolymer may form an amorphous precipitate or crystals suitable for X-ray diffraction analysis. Sometimes anything between these two extremes can be observed. Parameters such as pH and temperature, the chemical composition of the crystallization solution, and the rate of supersaturation decide whether an amorphous precipitate or crystals are formed. Supersaturation is often achieved by increasing the concentration of precipitating agent in the crystallization solution. Auxiliary substances may decide whether supersaturation leads to crystallization, or may improve crystallization.

Larger structures, such as the purple membrane¹³ or ribosomal particies¹⁴ may be grown into ordered two-dimensional structures in vitro. These structures can be investigated by optical or electron diffraction and subsequent three-dimensional image reconstruction, with 7 Å resolution.

Precipitation Reagents

The general mode of action of a precipitation reagent is the binding of water. This results in a water content insufficient to maintain the hydration of the biopolymers (or for complete protection of the biopolymers from one another). Salts, organic solvents and polymers are used as precipitants.

Salts

A rather limited set of salts has been used to produce protein and nucleic acid crystals. For most crystallizations it is essential to find the optimal salt concentration, which may be anywhere between 15 and 85% saturation. This value must be defined to a precision of 1-2%. Ammonium sulfate is the most widely used precipitant of the salt type. Citrate salts, due to their chelating properties, may be especially useful when the presence of divalent cations interferes with crystallization. Cetyltrimethylammonium salts are exceptional, since they increase the solubility of macromolecules in the crystallization solution with increasing concentration.

Organic Solvents

The optimal concentration of the organic solvent for the crystallization of a given biopolymer has to be ascertained with a precision of 1-2%, from a large concentration range, as in the case with salt type precipitants. In contrast to salt type precipitants, organic solvents can easily denature biopolymers. 2-methyl-2,4-pentanediol for example has been found to be a quite mild and efficient precipitant for otherwise sensitive macromolecules. 2-Methyl-2, 4-pentanediol, also known as MPD or hexylene glycol, is the most widely used precipitant of the organic solvent type.

Polymers

The most widely used polymers for crystallization are the polyethylene glycols. We have recently introduced PEG with the molecular weights 1000, 3000, 6000 and 8000 in the unique BioUltra quality.

Auxiliary Substances

Additional substances for crystallization may be of importance in more special cases. The presence of small polyamines, such as spermine, cadaverine, spermidine or putrescine, in the crystallization solution seems to be mandatory for the growth of high quality crystals of transfer RNA. These polyamines probably act as specific counterions for the negatively charged phosphate groups. X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA¹⁵ have clearly shown the potential of this method.¹⁶

With the exception of the concentration of the precipitant, the most important variable in the search for crystallization conditions is the pH value. A large variety of BioUltra buffers are available.

Since crystal growth generally requires time to reach completion, exposure to oxygen is likely to occur. It therefore may be wise to include a mild antioxidant (cf. section on "Antioxidants") such as cysteine, -mercaptoethanol, glutathione or dithiothreitol in the crystallization solution.

Metal ions may improve the crystallization of proteins irrespective of whether the ion is the cofactor of the apoprotein or not. Chelating agents, such as EDTA, have to be added to the crystallization solution if metal cations prevent crystal formation. Furthermore, a variety of small molecules and ions have been found to affect the crystallization process.¹

References

1. McPherson A. 1982. Preparation and Analysis of Protein Crystals. New York: John Wiley and Sons.

2. 1985. Methods Enzymol. 114, Sect II, 49 Elsevier:

3. Arakawa T, Timasheff SN. 1985. [3]Theory of protein solubility.49-77. https://doi.org/10.1016/0076-6879(85)14005-x

4. Feher G, Kam Z. 1985. [4]Nucleation and growth of protein crystals: General principles and assays.77-112. https://doi.org/10.1016/0076-6879(85)14006-1

5. Giegi R. 1987. Crystallography in Molecular Biology . p. 15. New York: Plenum Press.

6. Gilliland GL, Davies DR. 1984. [23] Protein crystallization: The growth of large-scale single crystals.370-381. https://doi.org/10.1016/s0076-6879(84)04104-5

7. Kim S, Quigley GJ. 1979. [1] Determination of a transfer RNA structure by crystallographic method.3-21. https://doi.org/10.1016/0076-6879(79)59070-3

8. DeLucas L, Bugg C. 1987. New directions in protein crystal growth. Trends in Biotechnology. 5(7):188-193. https://doi.org/10.1016/s0167-7799(87)80006-9

9. Henderson R, Unwin PNT. 1975. Three-dimensional model of purple membrane obtained by electron microscopy. Nature. 257(5521):28-32. https://doi.org/10.1038/257028a0

10. Arad T, Piefke J, Gewitz H, Romberg B, Glotz C, Müssig J, Yonath A, Wittmann H. 1987. The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures. Analytical Biochemistry. 167(1):113-117. https://doi.org/10.1016/0003-2697(87)90139-4

11. Kim et al. S. 1973. J.Mol.Biol.. 75421.

12. Ladner JE, Finch J, Klug A, Clark B. 1972. High-resolution X-ray diffraction studies on a pure species of transfer RNA. Journal of Molecular Biology. 72(1):99-101. https://doi.org/10.1016/0022-2836(72)90071-x