Protein Crystal Preparation

The growth of protein crystals is a difficnlt, complex, and often frustrating procedure. The protein crystal is precipitated from a snpersatnrated solntion of the macromolecule in which the protein is partitioned between the solid phase and the solntion. The pH value influences the solubility. Usually a pH is chosen near the isoelectric point of the macromolecule. Inorganic salts, organic solvents, and commercially available precipitating agents, such as the polymer PEG, can be helpful. 

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The ultimate in structural studies would, of course, involve X-ray crystallographic studies of protein crystals prepared from nonaqueous solvents of the kind that are now being so successfully carried out with certain protein crystals prepared from aqueous media (Kendrew et al., 1961). A priori, there is no reason to exclude the possibility that proteins might be crystallized from pure nonaqueous solvents, although no reports of such attempts have appeared. This is particularly so in view of the fact that in certain pure solvents, proteins appear to exhibit a more highly ordered (helical) conformation than they do in water solution. 

Oxford University Press, ISBN 0199636788 (paperback) T.L.Blundell and L.N.Johnson Protein Crystallisation, Academic Press, NY, 1976 A,McPherson Preparation and Analysis of Protein Crystals, J.Wiley Sons, NY, 1982 A.McPherson, Crystallisation of Biological Macromolecules, Cold Spring Harbour Laboratory Press, 2001 ISBN 0879696176.]... 

McPherson, A. The Preparation and Analysis of Protein Crystals. New York Wiley, 1982. 

A review on this topic was given by Finney in 1979154). As was pointed out by the author, at this time it was likely that at least some deviations of the hydration shell from that in solution would occur because there would probably be some remarkable perturbations of the hydration shell due to the interactions with neighboured molecules. Furthermore, the pH values and the salt concentrations necessary for preparing protein crystals are not identical with those under the usual conditions for native proteins in solution which could give rise to deviations in the hydration shell for crystallized and dissolved proteins. 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

McPherson A. 1982. Preparation and analysis of protein crystals. New York Wiley Interscience. 

As discussed below, crystallization of the enzyme is also an effeetive method for removing contaminating and denatured proteins. Crystallization has the potential to produce a preparation not only of high purity but also of extreme reproducibility in both composition and enzyme aetivity. An important property of crystallization is its inherent eapability to seleet for protein molecules that possess the same three dimensional strueture. This is in eontrast to other purification steps which are likely to induee some degree of denaturation. 

McPherson, A. (1982) Preparation and Analysis 402. of Protein Crystals, Wiley, New York... 

Such conclusions have been confirmed, in some cases, by X-ray crystallographic and NMR structure determinations. Although the CD conclusions, like those derived from IR and NMR studies, are for solution conformations, and the X-ray crystal structure must relate to the solid state, in fact X-ray measurements with proteins are usually carried out on a fragile crystal in which the molecule is bathed in solvent. Typically, a crystal prepared for X-ray work will contain 50% water by weight, so the crystal structure is effectively that of a molecule encased in, and thoroughly penetrated by, water. The aqueous solution conformation of the polypeptide is essentially the same as the conformation seen in its X-ray crystal structure. A representative example is egg lysozyme (Figure 2.6). 

An exception to the approach for determining protein solubility, that is based on concentrating the protein, occurs when the protein can be crystallized, in which case, the solubility of the protein is established by the protein concentration in the solution phase that is in equilibrium with the crystals. Though the crystals could also be dried and dissolved in other solvent systems to determine solubility in those systems, protein crystals carry salt and other ions, and may render different solubilities depending on the method of preparation. 

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