Protein crystallization temperature effects

Concluding this brief survey of the effects of cosolvents and temperatures on noncovalent binding forces between proteins, we may assume that while the dielectric constant may play a role in the cryoprotection of protein crystals, changes in interaction forces may confer protection or in some cases be responsible for crystal destruction. However, we must bear in mind that hydrogen bonds and salt links involved in the regions of contact between proteins will be strengthened and/or stabilized at low temperatures within certain limits of pan values, which should aid in the cryoprotection of protein crystals. 

E. Effects of Cosolvent and Temperature on Donnan and Electrostatic Parameters of Protein Crystals... 

In a study of a cytokine formulation, sulfates and a range of arginine salts were found to be effective in generating more than a 10-fold increase in solubility (Flores et ak, 2001). Unexpectedly, solubility increased with increasing sulfate concentrations, and the protein crystallized in sulfate formulations at low temperatures. These studies also indicated that counter ions of arginine with two or... 

Usually, the electron density around an atomic nucleus is temperature independent. However, large amplitude motions of atoms can yield a blurred picture of the density. Moreover, equivalent molecules in a protein crystal can adopt different conformations, an effect that can look similar to thermal motion. Explicitly identified conformers (and also holes , i.e. missing parts in some regions) are ascribed a certain occupancy, smaller than unity. Thermal effects and the remaining, non-explicitly represented conformational effects are usually treated in protein structure refinement implicitly by smearing out the atomic electron density using... 

Radiation damage and sample heating arise from the absorption of X-rays in the sample. The sensitivity of the specimen to these effects depends markedly on the temperature of the specimen. Initially comments will be restricted to room (or near room) temperature where protein crystals maintain their liquid-like nature in the solvent channels going through the crystal. 

At absolute zero the Bj are close to zero (equation (9.20)) and so are the thermal uj (equation 9.21)). However, for a protein crystal each unit cell is not exactly identical to the next, i.e. there is a statistical population of atomic coordinates causing an effective random disorder for all temperatures. Since the disorder is random equations (9.18) and (9.22) still apply and there are corresponding uj (equation (9.21)) due to the disorder. 

Unfortunately, no protein crystal exists with such a very small molecular disorder for the anomalously scattering atom and which is coolable to absolute zero if it did then the effective electron density for an anomalous/ or/" of only a few electrons would have an electron density considerably higher than the normal scattering electron density (by 50-100 times). In a practical case for metallo-proteins with a natural metal cofactor buried in the protein core, the disorder 5-factor will be considerably less than the overall disorder factor for the whole protein where the surface residues will have particularly large temperature factors. On the other hand, a heavy atom derivative bound to the surface of the protein will have a large disorder factor. 

On the whole, however, the usual relation— absorption of heat on solution and increase of solubility with temperature— is found much more commonly in protein systems, especially at low ionic strengths. Differ t proteins often differ markedly in heat of solution. The classical method of preparation of edestin involved its crystallization by the gradual cooling of a very warm salt solution of the protein. Marked differential effects of temperature on solubility have been observed for different components among the globulins of normal plasma, notably for y- obulin, and fibrinogen, and have been used already in certain procedures, especially for the purification of fibrinogen (Part VI). 

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

The freeze-drying process is initiated by the freezing of the biopharmaceutical product in its final product containers. As the temperature is decreased, ice crystals begin to form and grow. This results in an effective concentration of all the solutes present in the remaining liquid phase, including the protein and all added excipients. For example, the concentration of salts may increase to... 

As the temperature drops still lower, some of the solutes present may also crystallize, thus being effectively removed from the solution. In some cases, individual buffer constituents can crystallize out of solution at different temperatures. This will dramatically alter the pH values of the remaining solution and, in this way, can lead to protein inactivation. 

Finally, we note that all transfers to alcohol-water mixtures or additions of alcohol to crystal mother liquor involve changes in the proton activity of the solution. Care must be taken to ensure that the pH does not change too much, or the crystal may be disrupted. Worse still, the enzymatic activity may be abolished. Control of proton activity in mixed solvents is discussed in Section III,D. If dielectric effects are controlled and pH is properly adjusted, the microenvironment of a crystalline protein will correspond closely to that of aqueous solution at room temperature. Such correspondence is essential for temporal resolution of individual steps in a catalytic reaction. 

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