Chapter 12.1. The preparation of heavy-atom derivatives of protein crystals for use in multiple isomorphous replacement and anomalous scattering

Ligands may be classified as either hard or soft. Hard ligands tend to be electronegative and interact electrostatically, with little delocalization of electron density. Water molecules, glutamates, aspartates, terminal carboxylates, and hydroxyl groups of serine and threonine from the protein, as well as acetate and citrate ions from the buffer, fall into this category. Conversely, soft ligands are polarizable and tend to form covalent bonds. Typical examples include the anions Cl⁻, Br⁻, I⁻, S²⁻, CN⁻, imidazole, methionine, cysteine, cystine and histidine from the protein. Ligands can be listed in series of increasing hardness:

The metal components of the reagents may be classified as hard (class A) or soft (class B) in a similar way. Class A metals include the alkali metals, the alkaline earth metals, the lanthanide and actinide series, and the first-row transition metals from group III to group VA. Many of these metal ions have an inert-gas structure in which the electrons are held very strongly and tend to be non-polarizable. Metal ions in this class tend to interact with hard ligands, including the acetate, citrate and phosphate buffer components of mother liquor systems. On the other hand, class B metals have a preference for binding soft ligands. This group includes most members of the second and third row of the transition series (e.g. Ag, Cd, Pt, Au, Hg), which form cations such as or anions such as , and . The easily polarizable d electrons allow formation of covalent bonds with methionine, cysteine and imidazole, so displacing the ligands of the complexes. In the middle and towards the end of the first transition-metal series, the ions have properties intermediate between class A and B metals. Class B character increases in the series: Thus, zinc binds to the polarizable sulfur of cysteine and imidazole of histidine as well as to carboxylates and water molecules. Tl⁺ and Pb²⁺, which each have an inert pair of electrons in their outer shell, are stable cations and prefer carboxylate rather than sulfur ligands or imidazole.

The rates at which ligands enter and leave a metal complex are important in the formation of heavy-atom derivatives, especially the covalent complexes of mercury, gold and platinum. The rate-determining step in unimolecular S_N1 reactions is the expulsion of the leaving ligand from the metal complexes, which often proceeds relatively slowly. The intermediate complex, once formed, reacts with the entering ligand almost instantly. For S_N1 reactions, the rate is directly proportional to the intermediate complex concentration but independent of the ligand concentration. The bimolecular S_N2 mechanism involves attack by the ligand on the metal complex to form an intermediate complex, which then ejects the displaced ligand. The rate of reaction is proportional to the concentration of the initial species and the concentration of the nucleophile. S_N2 reaction rates are dependent on the nature of the leaving group and the attacking nucleophile in the following ways:

Sulfur ligands are good nucleophiles but poor leaving groups. They form thermodynamically stable complexes. The rate of leaving is influenced by the trans effect in square-planar complexes of Au(III) and Pt(II). Thus groups in square-planar complexes trans to NH₃ are difficult to displace. This has implications for attempts to make derivatives of proteins in ammonium sulfate, where ligands may be replaced by NH₃.

Rates of reaction depend not only upon which ligands are present in a heavy-atom complex but also on the character of the metal. For example, , and have similar square-planar geometries (Petsko et al., 1978), but the rates of substitution vary: Thus, if the reaction between the protein and a palladium or platinum complex is proceeding too fast, a gold derivative might be investigated.

In the environment of a living cell, the following oxidation states tend to be stable:

Although the pK_a of an individual amino acid in solution is generally defined within narrow limits, environmental and steric factors give rise to a wide range of values in proteins. Thus, the hydrogen-ion concentration influences the thermodynamic and kinetic stability of potential complexes. Protons compete with heavy-atom ions for the available binding site(s) on the protein. For example, below pH 3.5, cations bind less well to aspartic and glutamic acids due to the protonation of the carboxylate groups.

The nucleophilicity of histidine increases when it loses its proton, and thus its positive charge changes from around pH 6.0 to 7.0. Similarly, the nucleophilicity of cysteine increases dramatically when the thiolate ion is formed at pH ∼ 8.0. The thiolate ion is a stronger nucleophile than the thioether group of methionine, but when it becomes protonated it is considerably less effective. The nucleophilicity of the attacking groups varies in the order Thus the number and occupancy of sites can be manipulated by varying the pH, often after cross-linking the crystals to stabilize them.

Extremes in pH can give rise to considerable difficulties in establishing suitable derivatives, as hydrogen and hydroxyl ions compete with the metal ion/complex for the protein and with the protein for the metal ion/complex. At extremely high pH values metals in solution tend to form insoluble hydroxides. The ranges of pH values that are useful for metal ions are given in Table 12.1.3.1.

Varying the reactivity of amino-acid side chains by manipulation of the pH can enable the same heavy-atom ion/complex to bind at different sites, thus producing more than one derivative useful for phase determination.

Components present in the heavy-atom solution can have a profound effect on protein–heavy-atom interactions. The salting in/out agent (precipitant) and buffer are the principal sources of alternative ligands for the heavy-atom reagents, while protons compete with the heavy-atom ion/complex for the reactive amino-acid side chains.

Ammonium sulfate is the most successful precipitant in protein crystallization experiments (Gilliland et al., 1994). However, its continued presence in the mother liquor can cause problems by interfering with protein–heavy-atom interactions. At high hydrogen-ion concentrations, the NH₃ group is protonated (i.e. ), but as the pH rises the proton is lost, typically around pH 6.0–7.0, enabling the group to compete with the protein for the heavy-atom reagent by an S_N2 reaction.

The nucleophilic strength of potential ligands follows the order The anionic complex is present in excess ammonia at pH > 7.0 and it will react:

The resultant cationic complex is less susceptible to reaction due to the trans effect of NH₃. Pd, Au, Ag and Hg complexes react in a similar way. Decreasing the pH of the solution reduces the amount of free ammonia available through protonation (Sigler & Blow, 1965). Such a technique may give rise to other problems (e.g. cracked crystal, decreased nucleophilicity of the protein ligands).

Changing the precipitant to sodium/potassium phosphate or magnesium sulfate may alleviate the situation, but it may also present other problems. For instance, displaces Cl⁻ from , thus increasing the negative charge. Both and form insoluble complexes with class A metals (e.g. lanthanide and uranyl cations) (Petsko et al., 1978). Both acetate and citrate form complexes with class A metals, but citrate, a chelating ion, binds more strongly. Tris buffer is probably preferable; it binds many cations, but the complexes formed tend to be relatively unstable.

The solubility of a heavy-atom compound will depend upon the precipitant, buffer and pH. Typically, the component present in the highest concentration is the precipitant, either as salts (e.g. ammonium sulfate) or as an organic-based reagent (e.g. ethanol, MPD, PEG). Heavy-atom compounds that are essentially covalent and organic in character will be more soluble in ethanol, MPD, PEGs and other organic precipitants.

Although the solubility of tetrakis(acetoxymercurio)methane (TAMM) is higher than most multiple-heavy-atom compounds in aqueous solutions, the presence of glycylglycine or charged mercaptans, such as cysteamine or penicillamine, can increase solubility further (Lipka et al., 1976). The ratio of TAMM to solubilization agent (e.g. glycylglycine) is typically 1:10. Even so, the final solubility of TAMM depends on the concentration of competing anions (e.g. chloride) (O'Halloran et al., 1987).

Many organometallic compounds are relatively insoluble in aqueous solutions, but their solubility may be increased by pre-dissolving in an aprotic solvent such as acetonitrile.

Iodine and several inorganic iodide salts are insoluble in aqueous solutions. This can be rectified by dissolving the heavy-atom compounds in an aqueous solution of KI.

Most heavy-atom derivatives are prepared by diffusing or dialysing the compound into the crystals. Concentrations have typically ranged from 0.1–100.0 mM. Occasionally, concentrations as low as 0.001 mM have been employed to maintain crystal integrity. Low concentrations favour sites where the interactions between the heavy atom and the protein ligands are strongest. Decreasing the number of non-specific interactions minimizes the amount of heavy-atom reagent in the lattice. The latter absorbs X-rays without contributing to the diffraction pattern except at low angles. Increasing the concentration may give rise to other binding site(s). Usually, the higher the concentration employed, the shorter the soak time required for equivalent substitution. Short soak times at high concentrations tend to denature the crystals more often than long soaks at low concentrations. At very high concentrations (i.e. > 100 mM), the heavy-atom compound perturbs the protein crystal–mother liquor equilibrium by withdrawing water molecules from the hydration shell around the periphery of the crystal. Disorder of the crystals can sometimes be avoided by the application of a cross-linking reagent (e.g. glutaraldehyde). The optimal concentration is the lowest concentration that consistently reproduces intensity differences in the diffraction pattern of 15–25% without cracking and disordering the crystals.

Length of soak may be important. The heavy-atom data bank shows that, typically, soak times range from one day to one week. Useful derivatives have been prepared with a soak time of an hour to over a year. If no binding is apparent after several days, extending the soak time to over a week may produce some binding, but this is rare. Soaks of 24 hours for simple inorganic salts and up to one week for other types of heavy-atom compounds will normally suffice when screening for binding. The concentration of the heavy-metal compound that can be achieved will depend on its solubility in the crystal stabilization solution. Normally, the longer the soak, the greater the occupancy. Exceptions can arise due to undesirable chemical reactions between components present in the derivatization solution.

For covalent-bond formation, the length of soak and the concentration can be short (e.g. 1 h, 0.01 mM). This is especially true for mercury derivatives of proteins that have reactive sulfhydryls (Ringe et al., 1983).

Variations in the temperature can also alter the rate of reaction. The UO₂ acetate derivative of rhombohedral insulin binds twenty times more slowly at 4 °C than at ambient temperature (Blundell, 1968). A lower temperature allows greater control over the rate of substitution. Conversely, heavy-atom derivatives that do not appear to bind may do so upon elevation of the temperature.

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).