Section 12.1.5. Protein chemistry of heavy-atom reagents

Uranyl-ion complexes have proved the most popular A-group metal reagents for preparing heavy-atom derivatives of protein crystals (see Table 12.1.5.1). is a linear, covalent group based on U(VI), the most stable oxidation state of uranium. Table 12.1.5.2 lists the most commonly used uranyl derivatives. Uranyl compounds may show 2 + 4, 2 + 5, or 2 + 6 coordination, with ligands lying in or near a plane normal to the O=U=O²⁺ axis. These equatorial ligands may be neutral (e.g. H₂O) or anionic (e.g. , CH₃COO⁻,oxalate²⁻, F⁻, Cl¹ or ); the nitrate and acetate are bidentate ligands. An example is given in Fig. 12.1.5.1. Anionic complexes, such as , have been found near negatively charged amino-acid residues (e.g. Glu and Asp), suggesting that the equatorial ligands have been displaced. At low pH, uranyl groups have been located near the hydroxyl groups of threonine and serine residues.

Figure 12.1.5.1 | top | pdf | The binding site for uranyl ions in cytochrome b5 (oxidized: 3B5C). The positions of the ligands in the parent crystals are shown; these probably move in the complex.

The fifteen lanthanides have similar chemical properties and are generally used as nitrates, acetates or chlorides (Blundell & Johnson, 1976; Carvin, 1986). The lanthanide contraction, a steady decrease in size with increasing atomic number, allows selection of an ion with a radius that will give high occupancy and isomorphism. Gadolinium and samarium salts have the added advantage that the number of anomalous electrons is high.

Lanthanide ions have greater selectivity than the uranyl ion, which often forms clusters on the protein surface. Uranyl complexes and lanthanide ions are not very soluble above pH 7 and pH 9, respectively, due to the formation of hydroxides. Phosphate buffers should be avoided since they will compete for the heavy atom, often giving insoluble phosphates. In the presence of citrate, samarium is chelated and, since the citrate is difficult to replace, reaction may be inhibited. However, exchanging the buffer for Tris or acetate may enable a useful derivative to be obtained.

Thallium and lead can provide useful derivatives, especially in their lower oxidation states, Tl(I) and Pb(II), when they resemble class A metals. Owing to the non-group valence and presence of an inert pair of electrons, the ionic radii of Tl⁺ (1.44 Å) and Pb²⁺ (1.21 Å) are greater than most class A metals. Thallous and plumbous cations prefer carboxylate rather than imidazole or sulfur ligands, although Pb²⁺ occasionally manifests its intermediate character by interacting with imidazole groups. Thallic (Tl³⁺) and plumbic (Pb⁴⁺) ions are similar to class B metals, showing preferential binding to soft ligands, but they are easily reduced in protein solutions.

The most useful members of the B-metal group, platinum, gold and mercury, give rise to an extensive range of heavy-atom compounds which form covalent, electrostatic and van der Waals complexes with proteins. Some compounds can bind to the protein molecule in different ways; for example, can bind either covalently to the thioether group of methionine or electrostatically with positively charged residues.

Mercury compounds have proved very successful for preparing heavy-atom derivatives of protein crystals (Table 12.1.5.1), mainly due to the ease of formation of covalent bonds with cysteine residues. An example is given in Fig. 12.1.5.2 in which mercuric chloride has been used to replace zinc in thermolysin. Hg²⁺ complexes are commonly two-coordinate linear and four-coordinate tetrahedral. The most popular mercury reagents are given in Table 12.1.5.3. The covalent character in Hg—L bonds, especially in the two-coordinate complexes, can cause solubility problems in aqueous solutions. However, an excess of an alkali metal salt (e.g. ) will often convert the compound to a more soluble anionic complex of the type , where X = Cl⁻, Br⁻, I⁻, SCN⁻, NCS⁻, CN⁻, , oxalate²⁻, or . In the presence of ammonium salts at high pH values, the cationic tetraammine complex, , tends to form. Variation in the charge on the aromatic groups of organomercurials can give rise to different substitution patterns.

Figure 12.1.5.2 | top | pdf | Mercuric ions replace zinc in thermolysin (3TLN). The mercuric ion is shown superposed on the parent crystal structure; notice that the mercuric ion is slightly displaced from the zinc position due to its larger ionic radius.

Silver , used as the nitrate, tends to interact with cysteine or histidine (see Fig. 12.1.5.3). In the presence of ammonium sulfate, it probably reacts as the ammonia complex, . Silver ions are less polarizing and less reactive than Hg²⁺ ions; thus they give similar derivatives but often with less disorder, as in glucagon (Sasaki et al., 1975). Where the metal ion displaces a proton, Ag⁺ will need to react at a higher pH than Hg²⁺.

Figure 12.1.5.3 | top | pdf | The binding of a silver ion to immunoglobulin Fab (2FB4). The positions of the ligands in the parent crystals are shown, and these must move in the complex to coordinate the silver ion.

The class B metals palladium, platinum and gold form stable covalent complexes with soft ligands, such as chloride, bromide, iodide, ammonia, imidazole and sulfur groups. The stereochemistry of their complexes depends on the number of d electrons present. For instance, the d¹⁰ ion of Au(I) gives a linear coordination of two [e.g. ], whereas d⁸ ions of Pd(II), Pt(II) and Au(III) are predominantly square planar, giving cationic [e.g. ], anionic [e.g. , and ] or neutral [e.g. Pt(NH₃)₂Cl₂] complexes. These may accept an additional ligand to give square pyramidal coordination or two ligands to give octahedral coordination. The additional ligands are normally more weakly bound. Pt(IV) has a d⁶ configuration and forms stable octahedral complexes, such as , with six equivalent covalently bound ligands.

The kinetic and thermodynamic stability of these complexes depends on the protein ligands, buffer, pH and salting in/out agent (Petsko et al., 1978). Anionic groups do not readily react with anionic reagents, such as RS⁻, but are attacked more readily by neutral nucleophiles such as RSH, R-imidazole or RNH₂. The inert cationic group is most likely to form electrostatic complexes with anionic groups, such as carboxylate. The neutral Pt(NH₃)₂Cl₂ molecule, however, can penetrate into hydrophobic areas but requires a stronger nucleophile such as RS⁻. In acidic and neutral solutions, reacts most commonly with methionine (Figs. 12.1.5.4 and 12.1.5.5), cystine (disulfide) (Fig. 12.1.5.6), N-termini and histidine to form stable complexes. However, methionine reacts faster than histidine. Thus, it is possible to use time as a variable to define specificity. The most popular platinum reagents are listed in Table 12.1.5.4.

Figure 12.1.5.4 | top | pdf | The binding of through a methionine in azurin (1AZU).

Figure 12.1.5.5 | top | pdf | The relative positions of methionine side chains (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of . The methionine side chains have been least-squares fitted.

Figure 12.1.5.6 | top | pdf | The relative positions of cystine disulfide bridges (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of . The cystine side chains have been least-squares fitted, and only those with torsion angles in the range have been used.

In aqueous solution, the square-planar complex is hydrolysed to in about one hour, or in the presence of a protein, reduced to Au(I) by methionine. In ammonium sulfate it probably exists as AuCl₃(NH₃), and . In contrast, is more stable and normally binds electrostatically. However, on occasions at pH > 6.0, the complex has bound to cysteine residues by nucleophilic displacement reactions.

Osmium resembles platinum in many ways and typically acts as a class B metal. It occurs in all oxidation states from 0 to VIII, but most usually in III, as in K₃OsCl₆; in IV, as in K₂OsCl₆; in VI, as in K₂OsO₂(OH)₄; and in VIII, as in osmium tetraoxide, OsO₄. Higher-oxidation-state compounds tend to be reduced to OsO₂(OH)₂ in most crystallization solutions and in the presence of ammonia or halide ion they can become further reduced to cationic or anionic complexes, such as or . Anionic complexes may be substituted by histidine residues at pH > 7.0 or bound as ion pairs by histidine at pH < 7.0 or protonated amino groups. Cationic complexes tend to bind to negatively charged residues via electrostatic interactions.

Iridium is found in all oxidation states from II to VI but commonly exists in III, as in K₃IrCl₆, and IV, as in (NH₄)₂IrCl₆. Ir(III) is similar to rhodium(III) and is found in a variety of cationic, uncharged and anionic complexes. All Ir(III) complexes are kinetically inert, whereas most anionic complexes of Rh(III) are labile. Ir(IV) is commonly found as the hexahalo complexes (except iodine), which are also fairly kinetically inert. Cationic [e.g. ], neutral (i.e. IrCl₃) and anionic (i.e. ) species have proved useful in forming derivatives of protein crystals.

Positively charged groups of proteins, such as the α-amino terminus, ɛ-amino of lysine, guanidinium of arginine and imadazolium of histidine, may form ion pairs with heavy-atom anionic complexes. For example, and can bind through electrostatic interactions. Anionic metal cyanide complexes tend to be more resistant to substitution and consequently interact electrostatically on most occasions. For example, binds at several sites involving lysine or arginine residues in proteins (Fig. 12.1.5.7). and can act as inhibitors by binding at coenzyme phosphate sites.

Figure 12.1.5.7 | top | pdf | The binding of to aldose dehydrogenase (8ADH).

Since many heavy-atom reagents are hydrophilic, most interactions occur at the protein surface. However, substitution, addition or removal of the non-heavy-atom component(s) of the reagent can alter the hydrophilic–hydrophobic balance and lead to penetration of the core. For example, anionic complexes such as and are hydrophilic and would not normally enter the protein core, although organometallics, such as RHgCl and R₃PbCl (R = aliphatic or aromatic), are much more hydrophobic and can do so.

Hydrophobic organomercury compounds of the general formula RHgX, where R is an aliphatic or aromatic organic group, react with sulfhydryls through displacement of X. When X is , or , the bond is ionic, making the formation of the cation RHg⁺ easier. R is often chosen to be a small aliphatic group (e.g. CH₃, C₂H₅). However, the presence of a benzene ring enhances the stability of the heavy-atom reagent. Careful selection of the X group can assist penetration into the hydrophobic core. The hydrophobicity of X follows the order RHgR (R = aliphatic or aromatic) compounds also bind sulfhydryl residues in hydrophobic regions. The mechanism of reaction of methylphenylmercury with buried sulfhydryl groups may involve fast dissolution in the hydrophobic interior of the protein followed by a slow reaction with neighbouring sulfhydryl residues (Abraham et al., 1983). They are difficult to prepare in aqueous solutions; an aprotic solvent, such as acetonitrile, can improve solubility, but this is not normally a problem in high concentrations of organic components, such as PEG, MPD or ethanol.

Inert gases were first used in the analysis of myoglobin. Schoenborn et al. (1965) discovered that the hydrophobic site that bound also bound a xenon atom at 2.5 atmospheres. They proposed that this may be a general way of producing heavy-atom derivatives of proteins. Recently, there has been increasing interest in this idea, which has now been developed to produce well defined derivatives of a wide range of different proteins. Crystals are subjected to high gas pressures. Xenon requires about 10 atmospheres in order to get saturated binding sites. Krypton binds much less strongly and requires around 60 atmospheres. Since the binding of both inert gases is reversible, it is necessary to keep the protein crystals in a gaseous environment in a specialized pressure cell. Such pressure cells have been developed by Schiltz (1997) at LURE. Xenon binds to hydrophobic cavities, with little conformational change and a retention of isomorphism in crystals. Krypton binds at the same sites as xenon, but since it is lighter and needs higher pressure it has been exploited less by protein crystallographers. However, it has a well defined K edge at around 1 Å and so has attractions for multiple-wavelength anomalous dispersion.

In addition to their use in isomorphous replacement, iodine derivatives of crystalline proteins have been prepared as tyrosine or histidine markers to assist main-chain tracing and to act as a probe for surface residues. The order of reactivity towards these reactive residues is , I⁻, I⁺ and I₂ can be generated by several different methods. An equimolar solution of KI/I₂ or NaI/I₂ in 5% (v/v) ethanol/water solution is often used to generate the anionic species and I⁻. An oxidizing agent, such as chloramine T, can be added to KI, typically in a concentration ratio of 1:50; alternatively, polystyrene beads derivatized with N-chlorobenzene sulfonamide can be used with NaI. Similarly, the addition of excess KI to ICl or OI⁻ will generate , I⁻ and I⁺. To avoid oxidation of iodine solutions, the pH should be less than 5.0. To avoid cracking the crystals, it may be necessary to increase the iodine concentration very slowly and to wash the derivatized crystals in the mother liquor in order to remove free I₂. Mono- or di-iodination of tyrosines can cause disruption of the protein structure either because of the larger size or the breaking of hydrogen bonds due to lowering of the pK_a of the phenolic hydroxyl.

The structure determination of large multicomponent systems such as the 50S ribosomal subunit (Yonath et al., 1986) or the nucleosome core particle (O'Halloran et al., 1987) requires the addition of reagents with a greater number of electrons, preferably in a compact polynuclear structure. Such reagents may be either cluster compounds or multimetal centres having metal–metal bonds.

Polynuclear reagents should preferably be covalently bound to one or a few specific sites, either first in solution or later in the crystals. Spacers of differing length can be inserted into the reagent to increase accessibility. Their low solubility in aqueous solutions can often be overcome by dissolving them in an apolar solvent (e.g. acetonitrile). Tetrakis(acetoxymercurio)methane (TAMM) and di-m-iodobis(ethylenediamine)diplatinum(II) nitrate (PIP) have better solubility in aqueous solutions than other polynuclear heavy-atom compounds.

Polynuclear heavy-atom reagents give an enhanced signal-to-noise ratio in low-resolution MIR studies, but this advantage is offset by the fall-off in scattering amplitude that arises from interference of diffracted waves at higher resolution. In the nucleosome core particle, the scattering reached 50% of its zero-angle value at 7.0 Å, while the relative drop for a single heavy atom was 10% (O'Halloran et al., 1987). Cluster and multimetal reagents that have been successfully employed in protein structure determinations have been reviewed by Thygesen et al. (1996).