"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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    Enzymology 143-Gal. 14 Vol. 4 (4) 216 The value of /«• = 1.5 will not, of course, apply to a protein containing a substantial quantity of a nonprotein component, such as ferritin. In such cases more precise methods must be employed, and the density and nonprotein content of the crystals must be measured. The Implications of Protein Axes In considering the relationship between the molecule in solution and the molecule in the crystal the difference in the implication of screw axes and rotation axes should be realized. In a space group without rotation axes and with one molecule in the asymmetric unit there is no particular association of the molecules of ¿he cj\\s¡Uil into small groups. One would not expect, therQf'o^e, (to find the molecules in the mother liquoj; forming dimers, foj- .example. IÍ the space group contains a rotation axis, however, say a twofold axis, there is a very real sense in which the molecules of the structure *ire related in pairs, and it would not be »uprising if dimers wej-q found in the corresponding mother liquor. Insulin provides a very prejiy example of this. 12 ; 13 The usual crystals have the space group IW, which has a threefold axis, and under rather similar pll conditions the molecule is found to have a molecular weight not far from three times the molecu lar weight of the asymmetric unit of 12,000. At low pll different forms of the crystal are found, one of which has the space group P2|2i2i which contains only screw axes, and it is therefore not surprising that at low pi I the molecular weight in solution is found to be 12,000. Thus the space group of the crystal may give a very strong hint as to which condition in solution will show an unaggregated molecule. Notice here, as men tioned earlier, that the fact tlijit the asymmetric unit is 12,000 gives no * / V I C f I i Í ft evidence as to whether the true molecular weight of insulin is half this. Molecular Weight Determination It will be realized from what has been already said that the molecular weight as determined from the crystal alone may be a multiple of the true molecular weight and a multiple ora submultiple of the molecular weight found in solution. This fact will not be referred to again, and in » * ^ # * • \ * • * what follows the term molecular weight should be read as crystal molecular weight. A rough pallie of the molecular weight can be obtained fron] the volume of the dry unit cell by the empirical rule described earlier and with I: = 1.5. Note that this rule seems to apply irrespective of the amount of salt or alcohol in the wet crystal. This rule should be accurate to — 10 to +20% and will often be within —5 to +10%. There are, in fact, few other methods of molecular weight determination which will give a value as accurate as this from a single simple experiment. To obtain a better value it is necessary to measure the density of the crystal when it is in some well-defined state. This will allow the mass of the unit cell to be obtained from the observed volume. From this figure for the mass must be subtracted the mass of the nonprotein components of the unit cell—namely, the volatile material, such as water or alcohol, and the nonvolatile nonprotein material (such as salt) remaining after drying. The former is found by measuring the percentage loss of weight in taking the crystal from the original state (in which the density can be measured) to the vacuum-dried condition. The latter must then be found by chemical methods. What remains after subtracting these corrections is considered to be the molecular weight of the protein. There are three obvious choices for the well-defined state qf the crystal: the wet crystal, the air-dried crystal, an<j the vacuum-cjried crystal. Experience has shown 14 that the last of these is not suitable. The effect of vacuum drying is to remove the solvent from the spaces between the protein molecules and to leave holes. If the density of such a crystal is determined (by immersipn in a fluid), the fluid may enter these holes, and the observed density may not correspond to the density of the unit cell but be more nearly that of the protein molecule itself. One must therefore start from either the wet crystal or the air-dried crystal. At the moment of writing it is not clear which of these methods is the better for the enzymologist. In theory the wet crystal should yield a more accurate figure, but in practice the technique is more difficult, and this may perhaps lead to errors. On the other hand, the air-dried method can be carried out with rather small crystals, whereas the wet-crystal method requires large crystals. Both methods will therefore be described. They are followed by detailed accounts of the techniques of density determina tion and composition measurements. Air-Dried Crystals Air-dried crystals are not precisely defined for two reasons. First, the dimensions of the unit cell and the degree of disorder usually depend somewhat on the method of drying. Slow drying usually produces a more ordered crystal. Second, the cell dimensions and the water content vary with the humidity, though not very greatly— certainly much less than they do for wet crystals. Reasonable care should therefore be taken to keep the humidity the same for the different sets of measurements— on the cell dimensions, on the density, and on the nonprotein content. It is convenient to measure the cell dimensions first and to carry out the other measurements at the humidity of the atmosphere at the time oi the X-ray measurements. It should be not far from 40%. The rate of drying of a crystal can be reduced by obvious methods, such as restricting the entry of the dry air. A less obvious way is to immerse the wet crystals in xylene. 9 The water of the crystal is only slightly miscible with xylene, and because of this it diffuses out of the crystal at a very reduced rate. - II. Gutfreund, liiochcm ./. 60, 504 (1952). For a full discussion and references, see J. T. Kdsall, in The Proteins (Neurath and Bailey, eds.), Vol. 1, Part B, p. 717. Academic Press, New York; M>5:*. ^ !s B. \Y. Low and F. M. Richards, ./. Am. Chem. Sor. ui piuMfu 76 # ( 1 b +
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