Page 102 - Macromolecular Crystallography

ISOMORPHOUS REPLACEMENT 91

leucine, phenylalanine, proline, and valine. Argi- Cationic: This group includes heavy-atom
nine forms electrostatic interactions with anionic lig- reagents that predominantly bind to acidic regions
ands, while asparagine and glutamine may weakly on the protein through their overall positive
coordinate to simple metal complexes through their charge, such as (Pt(NH 3 ) 4 ) 2+ , (Ir(NH 3 ) 6 ) 3+ , and
side chain’s amide nitrogen. Like arginine, lysine (Hg(NH 3 ) 2 ) 2+ .
can form electrostatic interactions with anionic lig- Hydrophobic: This group includes the nobel gases
ands at pHs below its pKa. Near or above its pKa krypton and xenon, which bind to hydrophobic
of about 9, it can also react with platinum and pockets in the protein. The main impediment to
gold complexes. The indole ring nitrogen of trypto- the use of these gases has been the technical chal-
phan can be mercurated, but tryptophan is usually lenge in derivatization under pressure, particu-
buried in the protein and rarely accessible to sol- larly since pressurized capillaries of glass or quartz
vent. While the phenol hydroxyl oxygen of tyrosine are explosion hazards. A special device to make
is expected to be a good nucleophile, its pKa is nobel gas derivatives has been described by Schiltz
greater than 10. Thus, the tyrosine utility as a lig- et al. (1994), and a commercial one is now being
and for heavy atoms has been in the substitution of sold by Molecular Structure Corporation for use in
the phenolate hydroxyl with iodine (Sigler, 1970). cryocrystallography.
In addition to the side chains of amino acids, the Others: This includes iodine that can be used to
N-terminal amine and the C-terminal carboxylate mono- or di-iodinate tyrosine residues.
of a protein are potentially ligands for heavy-atom
derivatives.
6.5 Preparation of heavy-atom
Dr Bart Hazes (University of Alberta) has fur-
derivatives
ther grouped heavy-atom derivatives in six different
categories as follow: Macromolecular crystals grow in an equilibrium
state with their mother liquor. Disrupting this equi-
Class A: This class consists of the alkaline earth librium can often destroy the crystals or their ability
metals (Sr 2+ and Ba 2+ ), the lanthanides (La 3+ , to diffract X-rays. This situation can be exacerbated
Ce 3+ ,Pr 3+ ,Nd 3+ ,Sm 3+ ,Eu 3+ ,Gd 3+ ,Tb 3+ ,Dy 3+ , by the transfer of the crystal to a solution contain-
Ho 3+ ,Er 3+ ,Tm 3+ , and Yb 3+ ) and the actinide ing a heavy atom. Therefore, it is important, once
(UO 2 ) 2+ . As indicated above, these elements prefer crystals are removed from their sealed environment,
carboxylates and other oxygen containing ligands. to first transfer them to a stabilizing solution and
They withstand low pH and ammonium sulphate, let them re-equilibrate before further transfer to the
but they have lower solubility at higher pH and in heavy atom solution. Usually, a stabilizing solution
the presence of phosphates. is identical to the mother liquor in which the crys-
Class B: As described above, this group con- tal was grown, but with a higher concentration of
tains many of the most popular heavy-atom deriva- precipitant.
tives containing mercury and platinum, such as The mechanics of derivative preparation is
p-chloromercuribenzoate, HgC1 , mercuric acetate,
2 simple; it involves the transfer of one or more
ethylmercury chloride, K 2 PtC1 4 ,K 2 Pt(NO 2 ) 4 , and native crystals from the stabilizing solution to a
K 2 PtC1 6 . In general, these reagents prefer ligands solution differing only in the presence of a com-
containing sulphur and nitrogen such as cysteine pound containing the desired heavy atom. However,
and histidine. This group also consists of many before attempting to prepare derivatives, it is impor-
silver, gold, palladium, iridium, osmium, and cad- tant to recognize that heavy-atom reagents are very
mium containing reagents. toxic and must be handled with utmost care. These
Anionic: Thisgroupincludesheavy-atomreagents reagents are selected for their strong affinity for
that predominantly binds to basic regions on the biological molecules. Thus, they present real and
protein through their overall negative charge, such serious danger to their users. Once crystals have
−
as iodide, (HgI ) , (Pt(CN) 4 ) 2− , (IrC1 6 ) 3− , and been transferred to the heavy-atom solution, they
3
(Au(CN) 2 ) . can be soaked in that solution for a period of time.
−