Protein crystallization factors influencing

The most unpredictable process in X-ray structure determination is the crystallization of the candidate protein into a form suitable for X-ray diffraction. Each protein requires a unique set of conditions to form crystals. Typically 100 mg of highly purified protein is required to determine the conditions that result in usable crystals of 0.1 to 0.3 mm size, although a size of 0.3 to 0.8 mm is preferred. The occurrence of crystals and the rate of crystallization are influenced by many factors such as protein purity, the solvent, concentration of added precipitants, pH, temperature, and the presence of ions and cofactors. The protein solution at a concentration of typically 5 to 20 mg/ml is allowed to slowly reach supersaturation by the removal of or by changing the composition of the solvent by liquid-liquid diffusion or vapor diffusion methods. Microscale methods have been developed to explore several crystallization conditions simultaneously using minimum amounts of the purified protein sample. Recently, use of the zero gravity atmosphere in space has been explored as a means of facilitating crystallization (Eisenberg and Hill, 1990 Branden and Tooze, 1991 Tomasselli et al, 1991). 

Indeed, a direct relationship between the lifetimes of films and foams and the mechanical properties of the adsorption layers has been proven to exist [e.g. 13,39,61-63], A decrease in stability with the increase in surface viscosity and layer strength has been reported in some earlier works. The structural-mechanical factor in the various systems, for instance, in multilayer stratified films, protein systems, liquid crystals, could act in either directions it might stabilise or destabilise them. Hence, quantitative data about the effect of this factor on the kinetics of thinning, ability (or inability) to form equilibrium films, especially black films, response to the external local disturbances, etc. could be derived only when it is considered along with the other stabilising (kinetic and thermodynamic) factors. Similar quantitative relations have not been established yet. Evidence on this influence can be found in [e.g. 2,13,39,44,63-65]. 

As discussed previously, crystallization of multicomponent proteins is indispensable not only for determining the structure by X-ray crystallography but also for obtaining stable preparations at the high level of purity required for chemical composition and functional analyses. However, crystallization conditions are influenced by a multitude of factors, some of which will be described below. 

Table 2.4 presents some, but probably not all, physical, chemical, and biological variables that may influence to a greater or lesser extent the crystallization of proteins. The difficulty in properly arriving at a just assignment of importance for each factor is substantial for several reasons. Every protein is different in its properties, and surprisingly perhaps, this applies even to proteins that differ by no more than one or just a few amino acids. There are even cases where the identical protein prepared by different procedures or at different times show significant variations. In addition each factor may differ considerably in importance for individual proteins. 

The elution chart for impurities of crystals and mother liquid obtained from a Protein Column 1-125 are presented in Figure 4. There was no difference other than peak A between the pillar form and the thin plate form crystals. Although the amount of peak A detected in the mother liquid of the thin plate form crystals was almost the same as that in the mother liquid of the pillar form crystals, peak A was detected only in the thin plate form crystals. The location of peak A was close to that of L-phenylalanine, so this compound must have characteristics similar to those of L-phenylalanine. Thus, peak A might be one of the factors which affect the change from pillar form to thin plate form crystals. Future studies should be performed to identify the structure of peak A substance and clarify the influence of this substance on the change in the crystal appearance. 

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