Heavy-atom derivative protein

Figure 6.30 Representation in the Gaussian plane of the phase relationships derived by a single anomalous scattering atom in a heavy atom derivative protein. The structure factor of the protein vector, FP, lies on a circle of radius Fp centred at O. The structure factor of the heavy metal derivatives, FpH and FpH, lie on circles of radii FpH and FpH with centres at the tips of the vectors -Fpp and -FpH as drawn. The intersection of the three circles represents a unique solution to position of FP, corresponding to a single phase angle...

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. 

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. 

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. 

Figure 6.2 Vector (Argand) diagram showing the reiationships between heavy-atom derivative (Fpn), native protein (Fp) and heavy atom (Fpi) ap is the phase angie for the native protein. The vectors are piotted in the compiex piane.

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.

Since protein BLl 1 is nearly globular its location may be determined in a Patterson map with coefficients of [F(wild)-F(mutant)] and may serve, by itself, as a giant heavy-atom derivative. At preliminary stages of structure determination this approach may provide phase information and reveal the location of the lacking protein. 

In crystallography, heavy atom derivatives are required to solve the phase problem before electron density maps can be obtained from the diffraction patterns. In nmr, paramagnetic probes are required to provide structural parameters from the nmr spectrum. In other forms of spectroscopy a metal atom itself is often studied. Now many proteins contain metal atoms, but even these metal atoms may not be suitable for crystallographic or spectroscopic purposes. Thus isomorphous substitution has become of major importance in the study of proteins. Isomorphous substitution refers to the replacement of a given metal atom by another metal that has more convenient properties for physical study, or to the insertion of a series of metal atoms into a protein that in its natural state does not contain a metal. In each case it is hoped that the substitution is such that the structural and/or chemical properties are not significantly perturbed. 

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. 

Several diffraction criteria define a promising heavy-atom derivative. First, the derivative crystals must be isomorphic with native crystals. At the molecular level, this means that the heavy atom must not disturb crystal packing or the conformation of the protein. Unit-cell dimensions are quite sensitive to such disturbances, so heavy-atom derivatives whose unit-cell dimensions are the same as native crystals are probably isomorphous. The term isomorphous replacement comes from this criterion. 

Consider a single reflection of amplitude IFPI (Pfor protein) in the native data, and the corresponding reflection of amplitude IF pl (HP for heavy atom plus protein) in data from a heavy-atom derivative. Because the diffractive contributions of all atoms to a reflection are additive, the difference in amplitudes (IF,, pl — Fpl)... 

That is, the structure factor for the heavy-atom derivative is the vector sum of the structure factors for the protein alone and the heavy atom alone. 

In order to resolve the phase ambiguity from the first heavy-atom derivative, the second heavy atom must bind at a different site from the first. If two heavy atoms bind at the same site, the phases of will be the same in both cases, and both phase determinations will provide the same information. This is true because the phase of an atomic structure factor depends only on the location of the atom in the unit cell, and not on its identity (Chapter 5, Section III.A). In practice, it sometimes takes three or more heavy-atom derivatives to produce enough phase estimates to make the needed initial dent in the phase problem. Obtaining phases with two or more derivatives is called the method of multiple isomorphous replacement (MIR). This is the method by which most protein structures have been determined. 

As I described earlier, this entails extracting the relatively simple diffraction signature of the heavy atom from the far more complicated diffraction pattern of the heavy-atom derivative, and then solving a simpler "structure," that of one heavy atom (or a few) in the unit cell of the protein. The most powerful tool in determining the heavy-atom coordinates is a Fourier series called the Pattersonfunction P(u,v,w), a variation on the Fourier series used to compute p(x,y,z) from structure factors. The coordinates (u,v,w) locate a point in a Patterson map, in the same way that coordinates (x,y,z) locate a point in an electron-density map. The Patterson function or Patterson synthesis is a Fourier series without phases. The amplitude of each term is the square of one structure factor, which is proportional to the measured reflection intensity. Thus we can construct this series from intensity measurements, even though we have no phase information. Here is the Patterson function in general form... 

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. 

The metallointercalation reagents are a class of heavy metal derivatives that bind to double-stranded polynucleotides by inserting between adjacent base pairs in the helix.1 2 Prototype members of this class of intercalators are (2,2 6, 2"-terpyridine)(thiolato)platinum(II) complexes.3 These may be synthesized from chloro(2,2 6, 2"- terpyridine)platinum(II), which can both intercalate and bind covalently by losing chloride ion. Covalent binding of the thiolato complexes is much slower owing to the more inert character of the Pt—S bond. Metallointercalation reagents also have the potential to bind to proteins that have natural receptor sites for nucleic acid bases. They may therefore also be used to provide isomorphous heavy atom derivatives for X-ray analysis. 

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