Download Growing Gold-Containing Crystals of Biological Molecules Low S.M.1

Growing Gold-Containing Crystals of Biological Molecules
Low S.M.1
Department of Chemistry, Faculty of Science, National University of Singapore
10 Kent Ridge Road, Singapore 117546
ABSTRACT
Gold compounds are used clinically in the treatment of many diseases, such as rheumatoid
arthritis, cancer, malaria, asthma and AIDS. The knowledge of accurate molecular structures is a
prerequisite for rational drug design and to aid the development of effective therapeutic agents,
drugs and medicine. Crystallography can reliably provide the structure and atomic bonding details
of these molecules, which in turn may shed some light to the mechanism of these complexes. The
objective of this project is to isolate suitable single-crystals of gold-containing metallothionein,
which will subsequently allow full and unambiguous structure determination by single-crystal X-ray
diffraction techniques.
INTRODUCTION
Gold As a Therapeutic Agent
Chrysotherapy is the use of gold compounds in medicine. Gold (I) thiolates have been the
principle compound in chrysotherapy, the treatment of rheumatoid arthritis with gold-based drugs.
Chrysotherapy has an anti-inflammatory effect, retards and causes remissions of the disease state
and may contribute to the antimicrobial activity (Shaw, 1999). Besides rheumatoid arthritis, gold
complexes such as auranofin have been discovered to be effective against tumour activity.
A commonly cited reason for investigation of gold compounds relates the square-planar
geometry found in platinum in cisplatin. Gold (III) is iso-electronic with Pt (II) and forms similar
square-planar complexes. However, given that the mammalian environment is generally reducing
(because of proteins such as cytochrome which is found in the liver) compounds containing gold
(III) may be reduced in vivo to gold (I) and metallic gold.
The high affinity of gold (I) for sulphur and selenium ligands suggests that proteins, including
enzymes and transport proteins will be critical in vivo targets. The extracellular gold in the blood is
primarily protein bound, suggesting protein-mediated transport of gold during therapy (Shaw,
1999). Hence, metallothionein, being rich in cysteine residues and thus providing sites for the gold
to bind through the sulphur atoms, has great potential for further development as a therapeutic
agent.
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Student
Metallothionein
Metallothionein (MT) is involved in intracellular sequestration of metals (mainly zinc and
copper) including toxic metals (such as cadmium and mercury) in the body. MT are nonenzymic
low molecular weight proteins of extremely high sulphur and metal content. Of the 61 amino acids
in mammalian MT, 20 are cysteine and aromatic amino acids are absent. The covalent structure of
the MT is highly adapted to form metal-complexes. This property is linked to the presence of a
large number of cysteine residues and to their arrangement in the amino acid sequence to
accommodate the metal ions in tetrahedrally structured metal-thiolate clusters. All cysteine residues
participate in metal binding. There are no disulphide bonds and no labile sulphur. The most striking
structural feature in MT is the preservation of the cysteine residues, in which their positions are
identical in all mammalian MT.
MT is induced by zinc and copper and is responsible for the homeostasis of these metals, but
they also bind several other metals (including cadmium, silver, mercury and bismuth). Cadmium, a
toxic metal, also induces MT biosynthesis, so that formation of the Cd-MT complex plays and
important role in detoxification of the metal should they be present in the body (Harrison, 1985).
X-Ray Crystallography
X-ray crystallography is a combination of two independent subjects, crystallography and X-ray
diffraction. Crystallography deals with the arrangement of molecules inside a crystal. Crystals are 3dimensional ordered structures that can be described as a repetition of identical unit cells. The unit
cell is made up of the smallest possible volume that when repeated, is the representative of the
entire crystal.
X-ray diffraction focuses on the principles of diffraction of X-ray by a crystal to obtain structural
information. When X-rays are beamed at the crystal, electrons diffract the X-rays, which causes a
diffraction pattern. Using the mathematical Fourier transform, these patterns are converted into an
electron density map. Since electrons surround the atoms uniformly, it is possible to determine the
location of these atoms, and hence the molecular structure of the crystal may be obtained.
Crystals are formed when the conditions in a supersaturated solution slowly change. In this
project, the vapour diffusion method by hanging drop was used. In this method, a drop of protein
solution is suspended over a reservoir containing buffer and precipitant. As the concentration in the
reservoir is higher than that in the droplet, water diffuses from the drop to the solution. This
increases the protein and precipitant concentration in the drop and as vapour equilibrium is
achieved, the system is driven to supersaturation whereby the formation of crystals takes place.
EXPERIMENTAL SETUP
Preparation of Protein and Gold solution
As described (Melis et al., 1983), a 10mg/mL MT solution was prepared by dissolving 1mg of
MT II (from rabbit liver) in 100µL of solution containing 1.0M sodium formate and 0.2M
potassium phosphate at pH 7.5. The protein solution was then centrifuged and kept refrigerated (or
in an ice-box) at all times to preserve the protein.
In this project, we attempted to grow pure MT crystals and gold containing MT crystals. The
former was done by using the 10mg/mL protein solution prepared above as the hanging drop. The
latter was done by mixing an equivalent molar of the gold solution to the protein solution, i.e. 1.3µL
of Et3PAuCl solution in ethanol was mixed to 10µL of 10mg/mL MT solution. This mixture was
then subsequently used as the hanging drop for crystallization.
Crystallization
The vapour diffusion by hanging drop technique was used for crystallization. The reservoir was
filled with 1mL of the buffer and precipitant solution. 1µL of the protein solution was mixed with
an equal amount of reservoir solution, and was placed on a cover slip. The cover slip was then
inverted and placed over the reservoir. It was then carefully sealed with vacuum grease to prevent
evaporation. This was repeated with different precipitants and varied concentrations to find the
optimum conditions for crystallization.
The technique above was repeated using the MT-gold mixture as the hanging drop to obtain gold
containing MT crystals.
Data Collection
A crystal from Hampton Crystal Screen 1 condition-1 was frozen in liquid nitrogen and mounted
onto the X-ray diffraction machine to obtain the diffraction pattern.
RESULTS AND DISCUSSION
From the results obtained, only a few conditions gave crystals, needle clusters and crystal specs.
Using sodium formate as the precipitant, we were still unable to obtain the pure MT crystals. As
such, the concentrations of the sodium formate were varied (but maintaining the pH at 7.5) but the
crystals still proved to be elusive. As such, the precipitant was changed to ammonium sulphate
because of its high success rate as a precipitating agent for many protein crystals. Besides that,
PEG-3350, PEG-1500 and various crystal screens were used in search of the optimum condition for
crystallizing both pure MT and gold-containing MT crystals.
Needle clusters were found in almost every well which contained ammonium sulphate as the
precipitant, regardless of the concentration. These needles were far too small for any analysis or
determination. It is also highly skeptical that they are MT crystals for they were long and tetragonal
in shape, which is generally characteristic of salt crystals. The needle clusters may in fact be the
ammonium sulphate salt crystals themselves.
Polymers like PEG are long, unstructured and inflexible. They serve to dehydrate the protein
solutions, but at the same time they also crowd out other macromolecules from the solution,
depriving proteins of otherwise available solvent. This may explain why precipitate was found in all
the wells containing PEG as the precipitant.
Only crystals found in wells containing Crystal Screen condition-1 were suitable to be mounted
onto the X-ray diffraction machine. However, the diffraction pattern showed that they were salt
crystals instead of protein crystals. The crystals obtained here may be due to the sodium acetate or
calcium chloride salt, which is present in the crystal screen.
A stumbling block in this project was the insolubility of the gold complex in minimal volume of
solvent that would not affect the crystallization of the protein. This is because there is a need for
very pure protein in order to grow exceptionally good, well-ordered protein crystals. Therefore the
protein solution should only contain those chemicals necessary to maintain protein stability and at
the lowest concentration possible (McPherson, 1999). The three gold complexes provided were
Cy3PAuCl, Ph3PAuCl and Et3PAuCl. The possible solvents for these complexes are ethanol,
acetone, dichloromethane, chloroform and DMSO. However, only ethanol is suitable in the
presence of protein. In this light, only the Et3PAuCl complex was found to be soluble in ethanol,
and subsequently used in this project.
CONCLUSION
We did not manage to obtain any metallothionein or gold-containing metallothionein crystals.
This is due to the fact none of the conditions set up during the course of the project were the
optimum condition for the protein crystal.
In this project, we attempted to produce the gold-protein complex by mixing the protein and gold
solution in equivalent molar before crystallization. Had pure metallothionein crystals been obtained,
another method to produce the gold-protein crystals would be to soak the metallothionein crystals in
the gold solution. By soaking the pure protein crystal in the gold solution, there is a high possibility
for the gold atom to bind to the sulphur sites of the metallothionein during rearrangement and
further recrystallization of the complex. Thus a gold-protein complex crystal may be obtained.
In conclusion, the condition for crystallizing the metallothionein needs to be optimized further.
Conditions producing the first crystals or precipitate can be systematically refined by incrementally
varying each parameter such as pH, temperature, as well as precipitant and protein concentration.
ACKNOWLEDGEMENT
I would like to express my sincerest gratitude to Assoc Prof Tiekink and Dr Swaminathan for
their time and dedication in providing me this opportunity to work on this project. I would also like
to thank those in the IMA, DBS and Chemistry Lab for their help rendered during the course of this
project. Last but not least, many thanks to Marlin, my project partner, for working together with me.
REFERENCES
Harrison, P.M. (1985), Metalloproteins, Macmillan, NY.
Melis, K.A., Carter, D.C. and Stout C.D. (1983), ‘Single Crystals of Cadmium, Zinc
Metallothionein’, J. Biol. Chem., Vol 258, 6255-6257
McPherson, A. (1999), Crystallization of Biological Macromolecules, Cold Spring Harbor Press,
NJ.
Shaw, F. (1999), ‘Gold Based Therapeutic Agents’, Chem. Rev., Vol 99, 2589-2600