X-Ray Analysis and Protein Structure

Figure 31-3 Structure of the Hb subunit. Chains of amino acids in spiral or helical segments are linked by short, nonhelica segments.The helical segments are designated A through H. In this illustration, amino acids are designated in accordance with the helical or nonhelical segment in which they occur. (From D/ckerson RE. X-ray analysis and protein structure. In Neurath H, Ed. The Proteins composition, structure and function. 2nd ed. Vol. 2. New York Academic Press, 1964 603-778.)...

High resolution X-ray analysis of protein structures shows that the conformational categories of the connecting peptides which link the a-helices and -sheets are limited. Such well defined types of folding units, such as aa- and PP-hairpins, and aP- and Pa-arches, are referred to as supersecondary structures. One important step towards building a tertiary structure from secondary structures is to identify these supersecondary structure... 

Information concerning the tertiary structure of the proteins has been obtained from fluorometry, proton magnetic resonance spectroscopy, limited proteolysis, and X-ray analysis of protein crystals. 

X-ray analysis of proteins and nucleic acids is especially important as the absolute structure is needed for many advances in the field of medicine and biochemistry. 

In 1991, Luger et al. revealed by X-ray analysis the crystal structure of a natural DNA-histone complex. The X-ray structure shows in atomic detail how the histone protein octamer is assembled and how the base pairs of DNA are organized into a superhelix around it [74]. Since then this protein structure with cationic amino acids on the surface has acted as a model for the rational design of dendritic polymer-based gene vectors to mimic the globular shape of the natural histone complex [75-77]. 

In 1962, the Nobel Prize winners for Physiology and Medicine were Francis Crick, James Watson and Maurice Wilkins. They used chromatography to separate the complex mixture of amino acids making up proteins. This led to the characterization of the structure of a protein by X-ray analysis and in particular the realization that the three-dimensional structure of DNA was an inter-linked double helix. 

The first x-ray analysis of a protein, identifying the active site and giving details of secondary and tertiary structure, was that of lysozyme, carried out by Phillips [30] in 1966. The subsequent structure determinations of myoglobin [31] and haanoglobin [32] by Kendrew and Perutz represent great triumphs for both x-ray crystallography and protein science. 

Some modified human-lysozymes are designed and obtained by recombinant DNA technology. Some of them are crystalline proteins. The tertiary structures and their active sites have been investigated by means of X-ray analysis and molecular dynamics by computer-graphics as is shown in Figurer 5. 

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Biological fibers, such as can be formed by DNA and fibrous proteins, may contain crystallites of highly ordered molecules whose structure can in principle be solved to atomic resolution by x-ray crystallography. In practice, however, these crystallites are rarely as ordered as true crystals, and in order to locate individual atoms it is necessary to introduce stereochemical constraints in the x-ray analysis so that the structure can be refined by molecular modeling. 

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. 

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