Protein crystallization chemical modifications

The arrangements of the protein molecules in the crystals, i.e. the symmetries of the crystals, determine the patterns of diffraction, and the structures of the protein molecules are reflected in the intensities of all diffraction intensities. Therefore similar arrangements or structures of proteins molecules should eontribute similar diffraction patterns or intensities. In principle, from the known structures, the locations and translations of similar proteins inside different crystal cells can be deduced, and then the target proteins can be replaced by known model proteins to obtain initial models for the unknown crystal structures. This is the concept of molecular replacement. This method can be applied to proteins with similar structure for example, high sequence homologies, mutant proteins, proteins after chemical modification or substrate-binding protein. 

Virtually all of what is known about the secondary and tertiary structure of tubulin has been gleaned from a limited number of spectroscopic and chemical modification studies. Failure to obtain crystals of suitable quality for X-ray diffraction studies likely results from both heterogeneity in the subunits and the propensity of tubulin to polymerize into many polymorphs (see Section III). George et al. (1981) reported that as many as 17 distinct protein peaks may be discerned after isoelectric focusing of purified tubulin. 

There are essentially two types of control mechanisms for biochemical switching allosteric cooperative transition and reversible chemical modification. Allosteric cooperativity, which was discussed in Chapter 4, was discovered in 1965 by Jacques Monod, Jefferies Wyman, and Jean-Picrrc Changeux [143], and independently by Daniel Koshland, George Nemethy and David Filmer [116]. The molecular basis of this phenomenon, which is well understood in terms of three-dimensional protein crystal structures and protein-ligand interaction, is covered in every biochemistry textbook [147] as well as special treatises [215],... 

Figure 22 Introducing normative prosthetic group into metalloproteins. (a) by chemical modification of heme propionate. (Reprinted with permission fi-om Ref. 25. 2002 the American Chemical Society) (b) by noncovalent addition strategy. The crystal stmcture of the Fe (3,3-Me2-salophen) incorporated into AlalTGlaMb. (Reprinted with permission from Ref 287. 2004 the American Chemical Society) (c) by a single attachment strategy. The computer model of adipoc)4e lipid binding protein-phenanthroline complex. (Reprinted with permission from Ref. 291. 1997 the American Chemical Society) (d) by a dual covalent attachment strategy. The computer model of Mb(L72C/Y 103C) with a Mn "-Salen complex covalently attached at two-points and overlayed with heme. (Reprinted with permission from Ref 288. 2004 the American Chemical Society)...

When the crystal structure of a protein is not available, other techniques can be employed to identify the amino acids that are involved in its structure and function. Commonly used techniques include chemical cross-linking, site-specific chemical modifications, and mutagenesis. Chemical modifications of Met residues using oxidizing agents such as hydrogen peroxide, t-butyl hydroperoxide, chloramine T, and sodium periodate have been useful in identifying structure and function relationships in many proteins (1-7). 

For those proteins containing metal ions or metal clusters, such as Fe, Ni, and Cu, the MAD experiment can be carried out directly on the native protein crystals without the need of seleno-derivatives. Crystals containing heavy atoms incorporated via chemical modification or by soaking (McPherson, 1982) can also be used for MAD experiments. Finally, Dauter et al. (2000) has shown that ordered bromine in the protein s solvation shell could provide MAD phasing information. 

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