crystallization
crystallization
crystallization
One is faced with one of two possible starting conditions
1) no crystallization protocol exists, in which case one would be screening for possible crystallization conditions.
2) a crystallization protocol is in existence and therefore one would be trying to optimise conditions in order to improve either crystal size of quality.
In either case there are series of commonly used “industry standard” techniques which enable one to either screen or optimise protein crystallization conditions.
The most widely used methodology is that of vapour diffusion19, 46-47. In vapour diffusion, small droplets of precipitant solution and protein solution are mixed 1:1 on the surface of a glass coverslip (total volume of droplet is normally and approximately 10 µl) to provide a droplet that is in an initially undersaturated condition.
Chapter 1 Page 30 The coverslip is then inverted over a reservoir of solution which is at higher osmotic pressure than that of the droplet and the arrangement is sealed. Water is then drawn from the droplet by vapour diffusion until an osmotic equilibrium is struck between the droplet and the reservoir. In effect, this increases both protein and
precipitant concentrations slowly over a period of time until nucleation hopefully occurs (Figure 1.16). The droplet can be either in the ‘sitting’ or ‘hanging’ type configuration (Figure 1.14).
Figure 1.14 The sitting drop (left) and the hanging drop (right) vapour diffusion techniques for screening and optimization of protein crystallization trials. “[ppt]” in diagram refers to the concentration of
precipitant and is sometimes half that of the reservoir. Figures taken from ref.48
Another method based on vapour diffusion is the ‘sandwich’ technique49 (Figure 1.15). A protein and precipitant mix are placed between two siliconised glass coverslips and the droplet is dehydrated via vapour diffusion. This method allows ease of droplet visualization with microscopy, although the method is almost exactly like that of the standard sitting and hanging drop techniques.
Figure Figure Figure
Figure 1111....15151515 The sandwich drop vapour diffusion crystallization technique. The protein solution is sandwiched between a coverslip and water is drawn via vapour diffusion from the droplet by the reservoir.
Figure taken from ref.48.
Figure 1.16 Lysozyme/NaCl phase diagram taken from Iwai44 and co-workers. A typical path taken across
the phase diagram by a successful vapour diffusion experiment is shown by the series of black arrows. At position 1, the solution is undersaturated and is taken to position 2 where nucleation occurs. Due to crystal
nucleation, the local protein concentration decreases and the solution is taken to position 3 which is conducive to good crystal growth. Figure adapted from ref. 44
Chapter 1 Page 32 An infrequently used technique is that of free interface diffusion50 (Figure 1.17). In this method, protein solutions are placed in liquid contact with precipitant solutions within the confines of a narrow bore capillary tube. Both solutions are allowed to diffuse into one another thereby screening a wide variety of protein/precipitant combinations simultaneously across the length of the capillary. A related technique, called counter diffusion via the ‘gel acupuncture’ method51-52, allows diffusion of precipitant into ‘gelled’ (via the addition of agarose) high concentration protein solutions within narrow bore capillaries. The gelled protein solution can alternatively be layered with concentrated precipitant solution in a gel-slab type arrangement.
Figure 1.17 An example of a free-interface diffusion set-up. A narrow pore capillary is filled with both protein and precipitant solution and diffusion is allowed to occur. Figure taken from ref.48.
Figure 1.18 The microbatch method showing an oil droplet underneath a layer of oil. The green lines (bottom) indicate water diffusing out of the droplet. Figures taken from ref. 48.
In the microbatch method6, 53-56 (Figure 1.18 and explained in more detail further on), small volume droplets of protein and precipitant mix are placed beneath a layer of paraffin oil, silicon oil or a mixture of the two oils. Water can be drawn from the
submerged droplet by varying the ratio of silicon oil to paraffin oil, and this is called the modified microbatch method57-58. The degree of supersaturation and hence dehydration may therefore be altered by varying the ratio of silicon and paraffin oils under which the droplet is submerged60-61. Under non-dehydrating conditions, protein crystallizing solutions need to nucleate
as is
. This can sometimes be a major set back when using the microbatch method.Chapter 1 Page 34 An advantage of this technique is the ability to use small protein volumes. Additional advantage is gained by the lack of contact between the droplet and the container walls which can lead to unwanted heterogeneous nucleation57, 62-64.
Figure 1.19 A microdialysis arrangement showing a sample within a dialysis button sealed with dialysis membrane. Water and some precipitant are allowed to exchange between the button and the reservoir
across the membrane. Figures taken from ref. 48
Protein crystallization by dialysis59 (Figure 1.19) involves placing a protein sample in a dialysis button, which is then sealed with the aid of a dialysis membrane. Water and precipitant can exchange freely between the button and the surrounding reservoir through the membrane until an equilibrium is reached. With the dialysis technique one may alter the precipitant concentration whilst keeping the protein concentration
constant. Major disadvantages are that protein tends to get stuck to the membrane and is hard to remove.