Crystallization, of protein

Crystallization of proteins can be difficult to achieve and usually requires many different experiments varying a number of parameters, such as pH, temperature, protein concentration, and the nature of solvent and precipitant. Protein crystals contain large channels and holes filled with solvents, which can be used for diffusion of heavy metals into the crystals. The addition of heavy metals is necessary for the phase determination of the diffracted beams. 

James Sumner received the Nobel Prize for Chemistry in 1946 for the crystallization of proteins. Richard Willstatter, the 1915 Chemistry prizewinner, had proposed that proteins were not enzymes, and that the protein in urease was simply a scaffold for the veritable catalyst. Since urease is inactive without Ni, he was not so far wrong ... 

To determine the structures of drug compounds or protein molecules using X-ray crystallography, it is necessary to have these compounds or molecules available in crystalline form. For example, when crystals of protein are formed, the protein molecules are arranged in orderly fashions like tiny imaginary cubes stacked on top of each other. Each of these building blocks contains a molecule of protein and is termed a unit cell (Fig. 3.3). 

A major drawback with X-ray crystallography is the requirement to obtain crystals of proteins, which is a difficult process. However, techniques are being improved and many structures of proteins have been solved—more than 15,000 at the time of this writing. The very nature of crystallization also means that the protein molecules are frozen in space, rather than in the natural liquid state as found in the human body. 

Jancarik J, Kim SH. 1991. Sparse matrix sampling a screening method for crystallization of proteins. J Appl Cryst 24 409-411. 

Lenhoff, A. M., Pjura, P. E., Ddmore, J. G. and Godlewski, T. S. Jr. (1997). Ultracentrifugal crystallization of proteins transport-kinetic modelhng, and experimental behavior of catalase. J. Crust. Growth 180, 113-126. 

Jancarik, J., et al. (2004). Optimum solubihty (OS) screening an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Actu Crystallogr. D 60,1670-1673. 

Advances in computer design and improved speed of numerical iteration to make three-dimensional simulation of macromolecules practical without crystallization of proteins. These simulations provide structural information about active sites, binding domains, or immunodominant sites, and confirmation. Integration of these advances allow structure-based and computed-aided drug design. 

Crystal structure of a protein molecule can also be determined by x-ray crystallography. Purified protein is crystallized either by batch methods or vapor diffusion. X-rays are directed at a crystal of protein. The rays are scattered depending on the electron densities in different positions of a protein. Images are translated onto electron density maps and then analyzed computationally to construct a model of the protein. It is especially important for structure-based drug designs. 

Crystallographers grow crystals of proteins by slow, controlled precipitation from aqueous solution under conditions that do not denature the protein. A number of substances cause proteins to precipitate. Ionic compounds (salts) precipitate proteins by a process called "salting out." Organic solvents also cause precipitation, but they often interact with hydrophobic... 

Another vital type of ligand is a heavy-metal atom or ion. Crystals of protein/ heavy-metal complexes, often called heavy-atom derivatives, are usually needed in order to solve the phase problem mentioned in Chapter 2 (Section VI.F). I will show in Chapter 6 that, for the purpose of obtaining phases, it is crucial that heavy-atom derivatives possess the same unit-cell dimensions and symmetry, and the same protein conformation, as crystals of the pure protein, which in discussions of derivatives are called native crystals. So in most structure projects, the crystallographer must produce both native and derivative crystals under the same or very similar circumstances. 

The possibility of viewing both the image and the diffraction pattern is unique to electron crystallography and, in favorable cases, can allow phases to be determined directly. For an ordered array (a 2-D crystal) of proteins in a lipid membrane, the direct image is, even at the highest magnification, a featureless... 

First and foremost, cross-linked enzyme crystals are crystals. Within the crystal lattice the concentration of protein approaches the theoretical limit. This is important to the process development chemist, who would much rather use a small quantity of a very active catalyst in a reactor than fill it with an immobilized enzyme. Typically an immobilized enzyme contains only 1-10% by weight enzyme, with the remaining carrier material simply occupying valuable reactor space. The crystallinity is absolutely required to achieve the stability exhibited by CLCs [8], Cross-linked soluble thermolysin and cross-linked precipitate of thermolysin are no more stable than the soluble enzyme. Crystals of proteins... 

The best and most direct methods to observe the hydration of a macromolecule are X-ray and neutron diffraction analyses carried out at high resolution to better than 1.8 A. In the crystals of proteins, the macromolecules are heavily hydrated so that between 20Vo and 909o of the total volume are solvent. In fact, despite their well-defined crystal morphology, crystalline proteins more resemble concentrated protein solutions than the solid state. 

Salemme, F.R., A free interface diffusion technique for the crystallization of proteins for X-ray crystallography. Arch. Bio-chem. Biophys. 1972, 151. 

In many instances (cf. Cohn et al., 1947 King et al., 1956) proteins have been crystallized from mixtures of water and organic solvents, but to our knowledge, in no case from pure nonaqueous solvents. In this connection, the use of solvent additives to induce the crystallization of proteins involves the not fully recognized hazard that appreciable conformational changes may be induced in the protein molecules in such solvents (see Section 1Y,E). 

In view of the many experiments carried out in achieving the crystallization of proteins and subsequently refining those conditions to maximize crystal size and quality for X-ray structure determination, it is not surprising that examples of concomitant crystallization are found among proteins. We cite two here. Fu et al. (1994) reported the simultaneous crystallization of three polymorphs of an m-class glutathione... 

Membrane Proteins, Chemistry of Photoreceptors, Chemistry of Proton Translocation, Bioenergetics of Crystallization of Proteins Overview of Spectroscopic Techniques Overview of... 

Crystallization of Proteins Overview of Applications in Chemical Biology... 

Proteins, also, can vibrate in part or as a whole in the crystalline state. In contrast to crystals of small molecules, crystals of proteins almost invariably contain large numbers of molecules of solvent of crystallization, often corresponding to 50% or more of the unit cell volume. The extent to which these water molecules are ordered varies dramatically. Near the surface of the protein the water molecules may be well ordered. Beyond the first layer, water molecules typically show increasing levels of disorder. In addition, because of the high solvent content, there may be considerable motion and disorder in the protein molecule, particularly in the orientations of side chains. As a result, the number of independent Bragg reflections that can be measured is reduced, and this effectively reduces the resolution of the electron-density map. 

The osmotic second virial coefficient was used to examine the crystallization of proteins and their solubility in water and in aqueous mixed solvents. 

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. 

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