"X-ray diffraction of protein crystals"

Date:
1954-1956
Reference:
PP/CRI/H/1/33
Catalogue details

Licence: In copyright

Credit: "X-ray diffraction of protein crystals". Source: Wellcome Collection.

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    ö U H - b- sin ^8 Ê@ 1355 a Maple Press O RACK G GALLEY 186 Enzymology 143-Gal. 11 Voi. 4 (4) 216 |4] X-Ray Diffraction of Protein Crystals By V. H. C. C rick Introduction The X-ray study of protein crystals is a difficult and highly specialized field. Eventually, when the structure of a few proteins has been unraveled, the results will be of vital interest to enzymologists, since they should give information about the spacial arrangement of the active site of the enzyme. But meanwhile X-ray methods will have only a secondary interest for enzymologists. The equipment required for X-ray work is expensive and will nor mally not be available in an enzymological laboratory. This chapter has therefore been written on the assumption that the X-ray work will be carried out in collaboration with a professional crystallographer. It aims to show both the enzymologist and the X-ray crystallographer who may not be familiar with the X-ray work on proteins 12 -the kind ot information that can usefully be obtained in a fairly short time. A knowl edge of normal crystallographs terms and techniques 3 is therefore assumed, but all the special techniques required are described in detail. The most useful information that can be obtained from an X-ray examination of protein crystals is a rather good value for the molecular weight. If crystals of a reasonable size are available, a figure correct to a few per cent can be obtained in a few days. This method has been neg lected in the past, and enzymologists might well find it helpful. Occa sionally it can be shown that two proteins from different sources have very similar structures, and more detailed studies may, in favorable cases, yield some information on the shape of the molecule. Identification It might be thought that the very detailed X-ray picture oí a crystal won 1(1 \)C like a set of fingerprints and that it would provide a good jnethod of identifying a protein. This is not so, for two reasons. First, the same protein may form, under slightly different conditions, quite (liferent crystals, having radically different unit cells. Polymorphism is the rule rather than the exception in protein crystals. With presenj, techniques there is no method of deciding from the X-ray pattern thaf such different crystals contain the same protein. Second, proteins which are known to be similar, but are in fact different from the point of view of the protein chemist, can form identical crystals and give substantially identical X-ray pictures. 4 This is because the X-ray picture depends mainly on the broad architecture of the protein molecule, and small changes, such as the substitution of one amino acid for another, make such very slight changes to the X-ray intensities that in practice they can hardly be detected. It should be realized, however, that if two different proteins (e.g., from different sources) are found to give identical crystals, having very similar X-ray patterns, it is certain that the broad features of the struc ture of the two proteins are very similar. Such a case occurs, for example, among the myoglobins, in which it is found that crystals obtained from porpoise myoglobin and from three different species of whale give essen tially the same X-ray diagrams. 2 Thus in special circumstances protein crystals can give limited information about the identity of proteins. Crystallization Nothing will be said about the search for the conditions under which crystals can be pbtainpçl from amorphous protein, as the projein chemjsf js familiar with this problem, and there appear to be no general rules t(> act as guides. It will be assumed that crystals of some sort have already been obtained. Protein crystals must be a certain size, however, if they are to be studied conveniently by X-rays. The optimum is about 0.3 to 0.5 mm. in all directions, though preliminary work can sometimes be done on crystals as small as 0.1 mm., or even smaller. The protein chemist is usually satisfied if his crystals are large enough to be seen in the microscope, so it will often be necessary as a first step to grow larger crystals, especially as crystals as large as 1 mm. are required for very accurate molecular weight determination. There are many ways of doing this, but in essence they all consist in growing as few crystals as possible, and in growing these few rather slowly, so that fresh crystal nuclei cannot easily form. Proteins vary considerably: some may form quite large crystals with out any special precautions, whereas others produce such small crystals that considerable effort must be expended to get them large enough. An example of the first is chick lysozyme; with this enzyme from an amor phous precipitate large crystals will often form overnight. Trypsin inhibitor (from bovine pancreas), on the other hand, requires some weeks to form adequate crystals. The difference may depend on whether or not there is any marked difference in the solubility of the amorphous and the crystalline material. If fairly large quantities of the protein are available, it is simplest to make up a series of test tubes in which the protein concentration or the solvent composition is varied in very small steps from the conditions under which small crystals are formed. Those tubes, which initially show no crystals, may produce quite large crystals if left for a few days. If little protein is available a better method is to adjust the solvent so that from a concentrated protein solution a very slight precipitate is obtained. This should be carefully filtered to give a saturated solution free from crystal nuclei, and then very slowly concentrated further by evaporation. This can conveniently be done by placing a shallow layer of the solution, say a few millimeters thick, in the bottom of a fiat-bottomed tube. The tube is then sealed with a rubber bung containing a length of capillary (about 2 cm. long and 1 mm. internal diameter) to restrict the rate of evaporation, placed in a constant temperature desiccator, and left for some days or even weeks. If the crystals produced are too small the procedure should be repeated with a finer capillary tube or with a slightly less concentrated solution. Some workers favor the use of seed crystals. Large crystals of ribo- nuclease have been successfully grown by adding to the lyophilized protein powder a minute amount of crushed dried crystals to act as seeds and then adding solvent. The smaller the amount of seeds, the larger are the resulting crystals. Other methods are: the slow concentration of the protein solution by forcing it through a semipermeable membrane; a controlled and gradual change of temperature (if the solubility varies with temperature); or slow salting out by diffusing more salt into the solution through a semiperme able membrane (if alcohol forms part of the solvent, it. may be allowed to diffuse in through the vapor phase). It may even be possible to dispense with a membrane and merely place two layers of liquid (one of which contains the protein and the other alcohol or salts) one above the other, the lighter on top of the denser. Crystals may form on the walls of the test tube near the interface. 1 B. W. Ix>w, in The Proteins (Neurath and Hailev, eds.), Vol. 1, Part A, p. 235. Academic Press, New York, 1953. ('. Kendrcw, in *' Progress in Biophysics (Butler and Randall, eds.), Vol. 4, p. 211. Periamoli Press, London, 1951. a See, for example, C. \Y. Bunn, Chemical Crystallography. Oxford, New York, 1945. 4 M. F. Pcrutz, A. M. Liquori, and F. Iiirich, Nature 167, 929 (1951).
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