Proteins anomalous dispersion

MAD multiwavelength anomalous dispersion MAD -nine-substituted protein. 

Several structurally different types of HNLs occur in nature, which likely originated hy convergent evolution from different ancestral proteins. The enzyme from almond (PaHNL) was first crystallized in 1994 and the structure was solved by multiple wavelength anomalous dispersion of a mercury derivative. The first 3D structure analysis of PaHNL was performed in 2001. ° (7 )-PaHNL from almond uses FAD as cofactor and is related to oxidoreductases it exhibits HNL activity only in the oxidized form of FAD." ... 

Hendrickson, W. A., et al. (1988). Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation. Proteins 4, 77-88. 

Even if anomalous dispersion data are involved in the substructure determination process, it is the magnitude of the anomalous differences that are used, and the substructures of the biologically occurring macromolecule and its enantiomer are both consistent with the data. The probability that the substructure obtained by direct methods can be developed into a protein model with L-amino acids and right-handed a helices is 50%. Therefore, before proceeding further, other information must be used to determine the correct hand. 

The a vaiues are a measure of the electron-density variation in the protein and solvent regions, and the ratio of these numbers is a measure of the contrast between the two regions. Since anomalous dispersion data were used to phase the maps, the map for the correct hand will show greater contrast. In this case, the original direct-methods sites give rise to greater contrast thereby indicating that these sites do correspond to the correct enantiomorph. 

The X REL1 crystal structure, which consists of residues 52-316 corresponding to the N-terminal domain, was resolved using the SeMet multiple-wavelength anomalous dispersion method (9). The selenomethionines used for this method should be replaced with methionines to obtain the original form of the protein for simulations (see Note 2). 

Of course, I have just emphasized the so-called direct methods, which determine molecular structures ab initio from the observed intensities, and I have not even touched upon much larger macromolecular systems, such as proteins, which consist of thousands of atoms in the molecule. These structures also can be determined by special techniques. These include such techniques as multiple isomorphous replacement and anomalous dispersion, which take advantage of our ability to modify a given crystal containing molecules consisting of thousands of atoms by diffusing into the crystal a small number of really heavy atoms without disturbing the crystal structure. One then does the diffraction experiment on these so-called derivatives as well as on the native protein that one is interested in. 

This method of isomorphous replacement (Figure 8.27), together with anomalous dispersion data collection (see Chapter 14) is, to date, the principal method that has been successful for phase determination of macromolecules.Unfortunately, it is common to find that, although a heavy-atom solution has been soaked into a protein crystal, no regular (ordered) substitution has occurred, and solutions of other heavy-atom compounds must be tried. 

Some space groups are enantiomers of others. There are 11 such pairs, listed in Table 4.6 (Chapter 4) and Table 14.2. If the (+) isomer of a chiral molecule crystallizes in one of these space groups, the (-) isomer will crystallize in the enantiomorphous space group. The systematic absences in the Bragg reflections are the same for both members of these pairs of space groups, but anomalous dispersion can aid in distinguishing between them. Several proteins have crystallized in enantiomorphous space groups. 

The use of anomalous dispersion has led to many results of interest in chemistry and biochemistry such as the steric course of certain chemical reactions.The uses for detailed studies of chirality and for protein relative phase determination will now be discussed. 

Anomalous scattering can also be used directly if the protein is small and a suitable anomalous scatterer can be used. The three-dimensional structure of the small protein, crambin, was determined by W ayne A. Hendrickson and Martha Teeter by the use of anomalous dispersion measurements. This protein contains 45 amino acid residues and diffracts to 0.88 A resolution. It crystallizes with 72 water and four ethanol molecules per protein molecule. Since there is a sulfur atom in the protein molecule, the use of its anomalous scattering was made. The nearest absorption edge of sulfur lies at 5.02 A, but for Cu Ka radiation, wavelength 1.5418 A, values of A/ and A/" for sulfur are 0.3 and 0.557, respectively. Friedel-related pairs of reflections were measured to 1.5 A resolution, and sulfur atom positions were computed from difference Patterson maps. The structure is now fully refined and a portion of an a helix was shown in Figure 12.27 (Chapter 12). 

Anomalous dispersion measurements have proved to be very useful as an aid in phase determination in protein structure determination. 

The structure of PR-Toxin was established on the basis of the spectroscopic data of the parent metabolite and the changes to this data that arose as a result of simple reduction reactions. Its absolute configuration was established by anomalous dispersion in an X-ray structure. The toxicity has been associated with its inhibitory action on RNA and protein synthesis. 

The tunability of synchrotron radiation allows for data collection at or near the x-ray absorption edge of anomalous scatterers present in the protein or crystal to provide experimental phase information. Using techniques such as multi-wavelength anomalous dispersion (MAD) and single-wavelength anomalous diffraction (SAD) researchers are now able to solve macromolecular structures in a matter of days or weeks, a process that required months, or even years, a decade ago. 

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