The heavy-metal compounds used in crystallography are generally classified as either class A or class B (Blundell & Johnson, 1976
; Blundell & Jenkins, 1977
). Class A heavy-metal compounds, such as the lanthanides and actinides (primarily uranium), tend to bind to electronegative protein ligands through charge interactions, e.g.
binds to the carboxylate group of glutamate and aspartate, as seen in the heavy-atom-bound insulin structure (Blundell et al.
) and also in the prealbumin structure (Blake et al.
). In contrast, class B metals such as platinum, gold and mercury bind covalently to reactive amines and sulfhydryl groups (Islam et al.
; Rould, 1997
). However, other class B metals such as lead and thallium show a different reactivity and tend to interact with hydroxyl groups. Successful heavy-atom derivatization depends not only on the availability of specific amino-acid ligands in a given protein but also to a great extent on the crystallization conditions. Buffer and pH are known to affect the reactivity and solubility of heavy-atom compounds both through chelating heavy atoms and influencing the protonation state of the reactive groups.
To systematically assess the effect of buffer on heavy-atom reactivities, we carried out a series of derivatization experiments using peptides with a single reactive residue (e.g.
the methionine-containing peptide GEAGM
ASAGGAG) and class B heavy-metal compounds. These heavy-atom compounds generally form covalent adducts with amino-acid ligands and their reactivity depends less on the tertiary conformation of the ligands. Peptides with a single cysteine, methionine or histidine residue were assessed for reactivity with platinum, gold and mercury compounds, while peptides containing a single aspartate, glutamate, asparagine, glutamine or tyrosine residue were used in derivatization experiments with lead-containing compounds. A total of 43 heavy-atom compounds were tested for peptide reactivity in 12 buffer conditions over a wide range of pH. The results are tabulated in Agniswamy et al.
) and can be found at http://sis.niaid.nih.gov/cgi-bin/heavyatom_reactivity.cgi
. The database can be used to select compounds that are likely to derivatize a given protein of interest under selected buffer conditions.
As expected, heavy-metal compound reactivities depend strongly on buffer and pH conditions. Overall, MES and citrate buffers are the most and least supportive for heavy-atom derivatization experiments, respectively (Table 1). Therefore, proteins crystallized under MES buffer conditions are likely to be derivatized by a larger range of compounds than those crystallized in any other buffer. Among the basic pH buffers, reactions carried out in HEPES buffer have a greater success rate than those carried out in Tris buffers. However, depending on the peptide ligands available, heavy atoms may react preferentially in either HEPES or Tris buffer. The pH preference of heavy-metal reactivity is also apparent from this study. Gold potassium bromide, potassium tetrabromoaurate, gold potassium thiocyanide and trimethyllead acetate (TMLA) all show high levels of derivatization at slightly acidic to basic pH values, while potassium tetracyanoplatinate, gold sodium thiosulfate, mercury(II) chloride, methylmercury(II) bromide, p-chloromercuric benzoic acid, dichloroethylenediaminoplatinate and potassium hexachloroplatinate all react strongly under acidic conditions. It is interesting that K2IrCl6 and K2OsCl6 are observed to react consistently with the Met, Cys and His peptides in the vast majority of conditions examined, but the percentage of total peptide in a reaction which forms a heavy-atom adduct is consistently lower than that seen for other heavy-atom compounds.
List of the most reactive compounds for heavy-atom derivatization of proteins
Another observation which is clear from the data is that a number of compounds are highly reactive over a broad range of buffer and pH. The 22 most reactive compounds are listed in Table 1 and they include the seven compounds that were previously identified as highly successful in protein-derivatization experiments (Garman & Murray, 2003
; Boggon & Shapiro, 2000
). Other results that stand out include the observation that Met and Cys can be derivatized by at least four heavy-atom compounds in all buffers (Table 2). Methionine and histidine residues are the most reactive with platinum compounds, while cysteine preferentially reacts with mercury compounds. Thus, for proteins rich in methionine and histidine platinum compounds should be the first choice for screening, while mercury and gold compounds become the obvious candidates for proteins rich in free cysteines. Most importantly, the pH-dependent and buffer-dependent heavy-atom reactivity profiles enable the user to avoid experiments with compounds that are nonreactive in specific buffers, even in an ideal experimental scenario such as the heavy-atom peptide experiment carried out here.
Summary of peptide derivatization