Proteins, crystal structur heavy atom derivatives

Heavy-atom derivative of a protein The product of soaking a solution of the salt of a metal of high atomic number into a crystal of a protein. If the heavy-atom derivative is to be of use in structure determination, the heavy atom must be substituted in only one or two ordered positions per asymmetric unit. Then the method of isomorphous replacement can be used to determine the relative phase angles of the Bragg reflections. 

Multiple isomorphous replacement allows the ab initio determination of the phases for a new protein structure. Diffraction data are collected for crystals soaked with different heavy atoms. The scattering from these atoms dominates the diffraction pattern, and a direct calculation of the relative position of the heavy atoms is possible by a direct method known as the Patterson synthesis. If a number of heavy atom derivatives are available, and... 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

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. 

If two heavy-atom derivatives can be crystallized which preserve the space group and unit cell size of a large protein, then the structure can be solved directly this method of multiple isomorphous replacement was used by Perutz152 and Kendrew153 to solve the first two protein structures by laborious, decade-long film methods hemoglobin and myoglobin. 

X-ray structures are obtained from crystalline samples. Crystallization is a laborious process, and the crystallizability of a protein is unpredictable. Some proteins do not crystallize, and X-ray diffraction is not applicable in these cases. In other cases, heavy atom derivatives needed to solve the phase problem may be difficult to obtain. Furthermore, crystals, although highly hydrated, do not represent the structure of the molecule in a true solution. Movements of protein domains are restricted in the crystaUine state. NMR methods circumvent some of these difficulties because they are applied to proteins in solution. Therefore, in addition to permitting the determination of structures, the NMR technique is useful for revealing dynamic processes such as protein protein interactions. 

Isomorphous replacement is now employed in the determination of the structures of biological macromolecules. These molecules crystallize with 50% or more of the crystal volume filled with solvent molecules. Murray Vernon King, working with David Harker, conceived the idea of soaking protein crystals in solutions of compounds containing a heavy atom. These heavy-atom compounds are diffused into the crystals through the solvent channels and settle on preferred sites on protein molecules. The diffraction patterns of the unperturbed crystal (described as "native ) and the heavy-atom derivative are then compared in such a way that an electron-density map for the protein results. The method of isomorphous replacement, and the manner by which it is used to derive relative phases, are described in detail in Chapter 8. 

The MIR approach can be used to solve any protein structure de novo, but finding good heavy atom derivatives requires many crystals and is often a cumbersome and lengthy process. 

Aprotein crystal absorption is virtually eliminated for a typical crystal <1 mm in dimension. Sometimes it is not possible to make heavy atom derivatives owing to the chemical nature of a specific protein or the particular crystal form. Many proteins contain an essential metal atom or alternatively selenium can be incorporated into a protein. Similarly bromine can be incorporated into a nucleotide. In all these cases data can be collected at multiple wavelengths using SR and this allows phases to be determined. Protein structures have now been solved by several variants of these methods. This is an important technical capability because it either reduces the number of heavy atom derivatives that need to be found for isomorphous replacement or allows phase determination from a single crystal. 

The X-ray crystal structure of the photosynthetic reaction centre was the first membrane bound protein to be determined (Deisenhofer et al 1984, 1985). X-ray data were measured to 3 A for native and heavy atom derivatives with a conventional X-ray source and extended to 2.3 A using SR. 

In particular the structure determination of cucumber basic protein has been made using MAD data recorded on this instrument around the CuK edge (Guss et al 1988, section 9.7.5). This protein crystal structure had previously defied solution for many years because of problems associated with preparing heavy atom derivatives. 

Figure 17. Lead has been used as a heavy atom derivative to solve the phase problem in the crystal structures of a variety of protein including the calcium proteins calmodulin and synaptotagmin and die zinc protein 5-aminolevulinic acid dehydratase, ALAD. These structures provide useful insights into the coordination environments preferred by lead, which can bind both to carboxylate-rich calcium sites and thiol-rich zinc sites. Structures downloaded from die protein databank (3CLN, IRSY, 1AW5, IQNV) where necessary, lead was added to die figure based upon coordinates provided by the authors (240, 241, 243, 246). Figure adapted from Curr. Opin. Chem. Biol., Vol. 5, H.A. Godwin, The biological chemistry of lead, pp. 223-227, Copyright 2(X)1, with permission from Elsevier Science.

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