Preparation of heavy atom derivatives

Until recently, all protein crystal structures were solved by the method of heavy atom isomorphous replacement. The object is to introduce a heavy atom at one or a few sites on the protein such that the protein and the crystal lattice are perturbed as little as possible. The heavy atom acts as a marker for phase determination (Section 2(f)). Methods for the preparation of heavy atoms were comprehensively reviewed in reference 25. Here a summary is given, with brief notes on new developments. 

In metalloproteins, the metal cofactor may be removed and replaced by a heavier atom with similar chemistry. The zinc in insulin was successfully replaced by cadmium or lead, the zinc in carboxypeptidase and carbonic anhydrase by mercury, the zinc in thermolysin by lanthanide ions or strontium or barium and the calcium in staphylococcal nuclease by barium. Success has most usually been achieved by soaking the crystals in a chelating agent and subsequently diffusing in the heavier atom. 

Heavy atom-labelled inhibitors have the advantage that the specificity of the active site is exploited to generate a single site derivative. However, such reagents are likely to perturb the region of the enzyme of most interest. They have been useful in providing an approximate phase set that helps interpret a multi-site derivative and have then been discarded when the other derivatives are refined. 5-Iodouridine 2, 3 -phosphate was used in this way with ribonuclease-S [81]. 

Uranyl acetate which dissociates to in solution was first used successfully 

Platinum compounds have been widely used. These include the PtCl ion or the less reactive cis or trans platinum diaminodichloride compounds. At acid pH they react with methionine, cystine disulphides, N-termini or histidine. In the presence of ammonium sulphate, the chloride ions are rapidly substituted by ammonium ions to form [Pt(NH3)4] which is unreactive. Square planar negatively charged complexes such as Pt(CN)4 have been found to be effective in binding at the nucleotide-binding site in dehydrogenases. The cyanide ligands are firmly bound to the metal and are not displaced by protein atoms. 

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Hatfull, G. R, Sanderson, M. R., Freemont, P. S., Raccuia, P. R., Grindley, N. D. F. and Steitz, T. A. (1989). Preparation of heavy-atom derivatives using site-directed mutagenesis introduction of cysteine residues into yS resolvase. /. Mol. Biol. 208,661-667. 

X-ray crystallography is currently the most powerful analytical method by which three-dimensional structure information on biological macromolecules may be obtained at high resolution. Its application is however limited first by the preparation of single crystals suitable for X-ray diffraction and second by the so-called phase problem , that is the calculation of phases of difBaction data. Several approaches are available in order to circumvent this latter problem. The most commonly used methods are the multiple and single isomorphous replacement (MIR, SIR). These methods, as well as multiple anomalous difBaction (MAD), require the preparation of heavy atom derivatives, usually by the introduction of electron-dense atoms at distinct locations of the crystal lattice. This is usually done by crystal soaking experiments. 

Preparation of Heavy-atom Derivatives.— The problems of X-ray analysis of large proteins have also stimulated further study of the preparation of specific heavy-atom derivatives. Perham and Thomas have investigated the use of the imidoester methyl-3-mercaptopropionimidate. This reacts selectively with the amino-groups of proteins. The thiol group introduced can be used for the attachment of heavy-atom derivatives. There is a specific reaction of the imidoester with one of the two lysine residues of the protein subunit of intact tobacco mosaic virus (Scheme 1). However, the reagent... 

JT-Ray studies on the F a fragnients prepared by papain and pepsin fragmentation of human myeloma IgG immunoglobulins are being carried out by Poljak et /. The preparation of heavy-atom derivatives has involved considerable difficulties. A 6A electron-density map has been computed, which shows two clear domains (unpublished work) and is clearly of good quality. The data have been collected to 3.S A resolution, refinement of the heavy-atom positions of the derivatives has been completed, and an electron-density map computed. 

One of the earliest reports in which lead was used to prepare a heavy atom derivative was the crystal structure of the transfer RNA for phenylalanine (IRNA ) [Fig. 16(a)] (226, 227). This structure was a landmark in the field of RNA biochemistry both because it constituted the first crystal structure of a transfer RNA molecule (226) and because the observation that the Pb(II) soaked into the structure catalyzed cleavage of the sugar-phosphate backbone [Fig. 16(h)] helped to lay the foundation for the field of catalytic RNA (227). Although the observation that lead is able to catalyze autohydrolysis of RNA is widely cited as evidence that nucleic acids are a target for Pb(II), more detailed studies (228-238) suggest that the concentrations of lead required for this reaction are too high for this reaction to be physiologically relevant (see Section VI). 

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. 

Although the soaking method for heavy-atom derivative preparation is by far the simplest and most common, it is not the only method used. One can first derivatize the macromolecule, and then crystallize. This procedure is less frequently used because of drawbacks such as the inabihty to produce isomorphous crystals due to the disruption of intermolecular contacts by the heavy atoms. Other frequent problems are the introduction of additional heavy-atom sites (a potential complicating factor in phasing) by exposing sites hidden by crystal contacts, and changing the solubility of the derivatized macromolecule. 

Petsko, G. A. (1985). Preparation of isomorphous heavy-atom derivatives. Method Enzymol. 114,147-156. 

Heavy-atom derivation of an object as large as a ribosomal particle requires the use of extremely dense and ultraheavy compounds. Examples of such compounds are a) tetrakis(acetoxy-mercuri)methane (TAMM) which was the key heavy atom derivative in the structure determination of nucleosomes and the membrane reaction center and b) an undecagold cluster in which the gold core has a diameter of 8.2 A (Fig. 14 and in and ). Several variations of this cluster, modified with different ligands, have been prepared The cluster compounds, in which all the moieties R (Fig. 14) are amine or alcohol, are soluble in the crystallization solution of SOS subunits from H. marismortui. Thus, they could be used for soaking. Crystallographic data (to 18 A resolution) show isomorphous unit cell constants with observable differences in the intensity (Fig. 15). 

Final proof for the inferred structure (1) for cyclosporin A and a first insight in the shape of the molecule resulted from X-ray analysis and high-resolution NMR spectra. The preparation of a crystallized derivative containing a heavy atom was achieved by an addition reaction using iodine and thallium(I) acetate. Instead of the expected iodoacetoxy derivative, the cyclic product (11) was obtained. Obviously, the reaction proceeded by a selective addition of iodine to the double bond of the MeBmt unit followed by an internal cyclization with the participation of the OH group. Iodocyclosporin A (11) reverted easily with Zn powder in acetic acid into genuine cyclosporin A by rranr-elimination. X-ray analysis [6] revealed that iodocyclosporin A assumes a rather rigid backbone conformation. The amino acids 1-6 adopt an antiparallel, markedly twisted /i-pleated sheet conformation, whereas the residues 7-11 form a loop. 

Crystals of the material are grown, and isomorphous derivatives are prepared. (The derivatives differ from the parent structure by the addition of a small number of heavy atoms at fixed positions in each — or at least most — unit cells. The size and shape of the unit cells of the parent crystal and the derivatives must be the same, and the derivatization must not appreciably disturb the structure of the protein.) The relationship between the X-ray diffraction patterns of the native crystal and its derivatives provides information used to solve the phase problem. 

X 77.8 X 51.4 A, a space group of P2i2i2j, and a Z value of 4 (molecules per unit cell) (46). Several heavy atom derivatives, whose crystalline structure are isomorphous with that of the native enzyme, have been prepared for the X-ray diffraction analysis of crystalline cytochrome c peroxidase. 

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. 

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. 

The iodination of proteins has long been a very popular modification reaction. This reaction, performed under mild conditions, has been applied to such varied ends as the detection of side-chains at the catalytic sites of proteins, investigation of the relative reactivity of tyrosyl side-chains, and the introduction of heavy atoms in connection with X-ray diffraction studies. The availability of two radioactive iodine isotopes, 1 with a half-life of 56 days, and 1 with a half-life of 8 days, has been very extensively exploited in the preparation of hormone derivatives of high specific activity for radioimmunoassay and receptor studies. 

In our laboratory investigation a phase ambiguity arose when the experiment was performed only once. It was resolved simply by moving the oscillator to a new location and changing the phase of the reference wave. The analogous operation in X-ray crystallography is not quite so simple but still within reach. We must prepare a second (or even more, to be sure) heavy atom derivative bound at some other location in the unit cell. This second oscillator position generates then a second reference wave that yields, in the phase calculation, one phase solution in common with the first experiment. 

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