Heavy-atom derivatives of proteins

A number of studies have been performed with methyl picolinimidate (Benisek and Richards 1968 Plapp et al. 1971) aimed at exploring the usefulness of the metal-chelating properties of such derivatives in the preparation of isomorphous heavy atom derivatives of proteins for X-ray diffraction studies. 

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. 

Prepare heavy-atom derivatives of the protein with heavy atoms in different positions in the unit celt. Measure an intensity data set for each heavy-atom derivative. 

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. 

Soak complexes of heavy atoms of various kinds into a crystal. Screen diffraction data from heavy-atom derivatives of the protein for differences in intensities from those in the native data set. Calculate difference Patterson maps and locate the positions of the heavy atoms in the unit cell. 

FIGURE 9.11 The w = j plane of the difference Patterson map for the K2HgI4 heavy atom derivative of the hexagonal crystal form of the protein canavalin. The space group is P6, so w = is a Harker section. The derivative crystal contained two major K2HgI4 substitution sites and one minor substitution site per asymmetric unit. The Patterson peaks corresponding to those sites are marked with crosses. Note that the Patterson peak corresponding to the minor site cannot be discriminated from noise peaks in the Patterson map as is often the case. 

Figure 6a. A vector diagram illustrating the native protein (Fp) and heavy atom (F ) contributions to the structure factor (Fp,d for the heavy atom derivative of the protein. Sp, and are the phases for the native protein, the heavy atom, and the heavy atom derivative of the protein, respectively, b. The Marker construction for the phase calculation by the method of single isomorphous replacement corresponding to the situation shown in Figure 6a. The scale has been reduced slightly. The vector AB represents the amplitude (Fh) and phase (an) of the heavy atom. With centre A a circle radius Fp is drawn. Similarly, with centre B a circle radius Fp is drawn. The intersections of the circles at O and O represent the two possibilities for ap. Only one (O) is the correct solution.

Figure 7. A vector diagram illustrating the effects of heavy atom anomalous scattering on the reflections hkl (denoted +) and filci (denoted —). Fpn is the average structure factor amplitude for the heavy atom derivative of the protein. FJ Ih imaginary part of the heavy atom structure factor amplitude which arises from anomalous scattering. Because FJl always advances the phase by fI/2, FpH (+) and Fpn (—) are no longer equal. The measured difference between these amplitudes can be used fbr phase determination.

The first protein structures were derived using a technique called isomorphous replacement (IR), developed in the late 1950 s. The materials used are heavy metal derivatives of protein crystals. To obtain a heavy metal derivative of a protein, the protein crystal is soaked in a solution of a heavy metal salt. The metals most used are Pt, Hg, U, lanthanides, Au, Pb, Ag and Ir. The heavy metal or a small molecule containing the heavy metal, depending upon the conditions used, diffuses into the crystal via channels created by the disordered solvent present. The aim is for the heavy metal to interact with some surface atoms on the protein, without altering the protein structure. This is never exactly achieved, but in suitable cases, the changes in structure are slight. 

Figure 6.26 Representation in the Gaussian plane of the phase relationships between the structure factor of a pure protein, FP, a heavy atom, FH and an isomorphic heavy atom derivative of the protein,...

Use of Lead as a Heavy Atom Derivative in Proteins and Nucleic Acids... 

Patterson maps and direct methods are generally successful for structures containing fewer than 50 atoms per asymmetric unit. They sometimes work for larger structures like vitamin B 12 as well. For proteins and other really large structures, however, the most successful method is that of chemical substitution. The phase problem is solved by comparing the X-ray patterns for two heavy-atom derivatives of the biological molecule. Fourier analysis then gives the shape of the molecule. 

Referring to figure Bl.8.5 the radii of the tliree circles are the magnitudes of the observed structure amplitudes of a reflection from the native protein, and of the same reflection from two heavy-atom derivatives, dl and d2- We assume that we have been able to detemiine the heavy-atom positions in the derivatives and hl and h2 are the calculated heavy-atom contributions to the structure amplitudes of the derivatives. The centres of the derivative circles are at points - hl and - h2 in the complex plane, and the three circles intersect at one point, which is therefore the complex value of The phases for as many reflections as possible can then be... 

Figure Bl.8.5. Pp Pdl and Fdl are the measured stnicture amplitudes of a reflection from a native protein and from two heavy-atom derivatives. and are the heavy atom contributions. The pomt at which the tliree circles intersect is the complex value of F. ...

Figure 18.10 The diffracted waves from the protein part (ted) and from the heavy metals (green) interfere with each other in crystals of a heavy-atom derivative. If this interference is positive as illustrated in (a), the intensity of the spot from the heavy-atom derivative (blue) crystal will be stronger than that of the protein (red) alone (larger amplitude). If the interference is negative as in (b). the reverse is true (smaller amplitude).

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... 

However it would be a great mistake to believe that all actions of metal drugs will be at the DNA level. The metal complexes described here act in a highly selective manner with proteins — this is why they are used to provide heavy atom derivatives for crystallographic work. Thus we may expect that there will be other effects of the heavy metals which are associated with RNA and protein interactions. 

The isomorphous replacement method requires attachment of heavy atoms to protein molecules in the crystal. In this method, atoms of high atomic number are attached to the protein, and the coordinates of these heavy atoms in the unit cell are determined. The X-ray diffraction pattern of both the native protein and its heavy atom derivative(s) are determined. Application of the so-called Patterson function determines the heavy atom coordinates. Following the refinement of heavy atom parameters, the calculation of protein phase angles proceeds. In the final step the electron density of the protein is calculated. 

NCP crystals. There were two facets to this approach. First, it was necessary to reconstitute NCPs from a defined sequence DNA that phased precisely on the histone core to circumvent the random sequence disorder. It was obvious that the DNA was important for the quality of the diffraction from NCP crystals but the role of histone heterogeneity was not so clear. Heavy atom derivatives (i.e., electron rich elements bound in specific positions on the proteins) were not readily prepared by standard soaking experiments, due to a paucity of binding sites. Hence, it was necessary to selectively mutate amino acid residues in the histones to create binding sites for heavy atoms. 

From Eq. 3 and Fig. 6.3a it is clear that with only one heavy-atom derivative (single isomor-phous replacement SIR) the resultant phase will have two values (ap and apb) one of these phases will represent that of one structure and the other of its mirror image. But, since proteins contain only L-amino acids, this phase ambiguity must be eliminated using a second derivative, the anomalous component of the heavy atom or by solvent levelling (Wang, 1985), as shown diagrammatically in Fig. 6.3b. 

Figure 6.3 Isomorphous replacement phase determination (Marker construction), (a) Single isomorphous replacement. The circle with radius Fpp represents the heavy-atom derivative, while that with radius Fp represents the native protein. Note that the circles intersect at two points causing an ambiguity in the phase angle apg and apt, represent the two possible values, (b) Double isomorphous replacement. The same construction as that in single isomorphous replacement except that an additional circle with radius Fpn2 (vector not shown for simplicity) has been added to represent a second heavy-atom derivative. Note that all three circles (in the absence of errors) intersect at one point thus eliminating the ambiguity in the protein phase angle ap. Fm and Ppy represent the heavy-atom vectors for their respective derivatives.

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