Biotechnology

Access to synchrotron light is becoming increasingly important for researchers developing industrial applications of biotechnology, in such areas as bioremediation and biological sensors. Enzymes to degrade industrial and environmental pollutants are being designed to alter their substrate specificity by modification of the active sites of natural enzymes that degrade similar chemical moieties, by protein engineering based on the three dimensional structure of the native enzyme. These modified enzymes themselves or the genes that code for them can be inserted into biological organisms that can be used in the remediation of contaminated environments. These techniques can also be applied to the removal of toxic metals and the concentration of metals from low-grade ores. The engineering of the thermostability of enzymes used for industrial purposes at various temperature regimes can be carried based on structural information.

The design of insecticides with increased efficacy and species specificity is also being investigated through structural biology. The new classes of insecticides target insect hormone receptors, enabling the disruption of normal insect growth, by the design of agonists or antagonists to modulate the function of these receptors. Such biotechnological applications can have an enormous impact on the environment and the rural sector, for example by the control of specific insect pests in agriculture and livestock industries.

Structural biology

Tremendous strides have been taken over the past twenty years in the understanding of biology and the processes that make life possible. Central to this has been the understanding of the role of genes, and the breaking of the DNA code. Recent unravelling of the human genome had led to the perception that this knowledge will help cure many intractable diseases, and the control and manipulation of biota is possible

Understanding the genome is important, but of greater interest in the post-genomic era is an understanding of the structure and function of the many proteins that are expressed by the gene. This is a very large undertaking-it is estimated that there are over one million different products compared to the 30000 genes that make up the human genome.

Thus a new field of proteomics has emerged that involves the systematic characterization of the gene products of entire organisms. Beyond proteins, there are other complex macromolecules of key importance in biological processes, such as viruses and nucleic acids.

The determination of the three dimensional structures of these complex macromolecules is known as structural biology. Perhaps the best-known technique employed by structural biologists is protein crystallography using single crystal x-ray diffraction, which provides the primary structural information; the crystal structure of the molecule. But knowledge of the crystal structure alone is insufficient; of equal and sometimes greater importance is the elucidation of the shape of the molecule and how it is folded. This is the secondary, or conformational, structure of the molecule.

Although nuclear magnetic resonance (NMR), mass spectrometry, and cryo-electron microscopy do provide valuable information on the primary and secondary structures of complex macromolecules, x-ray diffraction, small-angle scattering and circular dichroism are the key techniques used in structural biology.

Early attempts to analyse macromolecular structures used conventional x-ray and laboratory light sources; the most famous of these is probably the determination of the structure of DNA by Watson and Crick. However, it was not until developments over the past decade in cloning and over-expression of proteins, more effective methods of protein manipulation, cryogenic cooling of the crystals to minimize degradation by the radiating beam, advances in computer and detector technology and, in particular, access to synchrotron light that large-scale structural biology has been possible.

The growth in activity has been spectacular. A measure of this growth is the number of structures registered in the Protein Data Bank over the period spanning 1972 to 2002. The remarkable rise after 1992, in particular, coincides with the commissioning of a number of synchrotron-based protein crystallography beamlines. At 7 October 2003, the number of proteins registered in the bank was 22810, of which 19380 were determined by x-ray diffraction.

Australia has been a major player in proteomics-the term was coined by researchers at Macquarie University. Access to the protein crystallography, SAXS/WAXS and circular dichroism beamlines at the Australian Synchrotron will maintain the world-leading position of Australian researchers in this area.

Protein Crystallography

Macromolecules

Small molecules

A second end-station at the Australian Synchrotron will be dedicated to the study of small molecule and macromolecular structures to resolutions from normal to high (3 to 0.1 Angstrom) on weakly diffracting crystals and on crystals for charge density studies.

The use of the brilliant, highly collimated and tunable x-ray radiation from a synchrotron will enable structural studies that are impossible with conventional laboratory sources, or will provide vastly improved precision and accuracy than that presently available within Australian laboratories. Unlike any other radiation source, synchrotron light is tunable and this will provide contrast between isoelectronic species and also sensitively discriminate between oxidation states.

Applications of the small molecule facility will include structure determination on microcrystals with dimensions of a few micron or less. No other technique has the ability to assign unambiguously atom-to-atom connectivity, stereochemistry, absolute configuration and charge density distributions.

Moreover, the facility will allow the resolution of disorder, offering an insight into its relation with physical properties, the identification of superstructure and structures under change, such as pressure. This will enable the Australian research community to elucidate the structures of an ever-increasingly complex selection of molecules and materials, for which it is difficult to grow large crystals for conventional x-ray studies.

Biosystems

The electronic properties and interactions of matter at an atomic level in biological environments is very much unknown. Yet the detailed understanding of these systems is crucial to the successful development of many new technologies that have direct impact on biosystems. These include medical implants, delivery systems such as those required for radiopharmaceuticals, bio-sensors and chips for diagnostics, biomimetic materials such as the construction of artificial skin and organs, and novel photosynthetic devices. Vacuum ultraviolet spectroscopy (VUV) is able to probe the valence and low-lying core states of many elements in the periodic table. The interaction of such states ultimately controls the complex interactions and properties observed in biological systems.

The high flux and small spot size produced by VUV synchrotron light sources will allow for many ground-breaking experiments and studies to be performed on biosystems from a sub-angstrom to micron scale. Studies will initially focus on more traditional, but still poorly understood, systems such as the electronic properties of amino acids on various surfaces. A significant new direction would be the in situ study of liquid-solid interfaces and sandwiches. As most biosystems are made of several functioning parts, small spot microscopy on objects as tiny as only a few nanometre to as large as a few micron in size would be of tremendous importance in order to determine accurately the electronic state of each component part of the system.

Surface science, which is supported by soft x-ray, vacuum ultraviolet and vibrational spectroscopy techniques, has recently been increasing in prominence in the biomedical area, based on the fact that many biological reactions occur at surfaces. Any fundamental understanding of the biocompatibility of a medical device must take into account the properties of proteins and cells at interfaces, and the characteristics of local biological reactions. Principles worked out in surface science laboratories are likely to become the basis for ways of improving the function and durability of materials featured in a wide range of medical products.

As an example, the hemocompatibility of synthetic surfaces can be improved by various biologically active substances, of which heparin is perhaps the most promising. To immobilize heparin onto biomaterial surfaces, its physicochemical properties are modified by incorporation of a specific binding agent onto the heparin molecules. The resulting modified-heparin coating material has a high affinity for a variety of synthetic surfaces, and retains all biological properties of the unmodified heparin. This offers the prospect, for example, of heparin-coated bypass circuits for use in open-heart surgery.

Rational Drug Design

Many medicines have been developed by traditional drug discovery methods, in which a myriad of naturally occurring compounds have been surveyed for their ability to control disease. However, the explosion in the fundamental knowledge of biological protein interactions has enabled a rational approach to drug design based on theory and structural biology. The impact pf structural biology on the design of medically important drugs is exemplified by the development of the anti-influenza drug Relenza. This work was carried out within CSIRO and was the first structure-based anti-viral drug that was developed, and also a very early example of the rationally-based drug design methodologies. Subsequently, the new generation of drugs active against HIV such as HIV-protease inhibitors were developed by a similar methodology. Other examples have been the development of the anti-inflammatory inhibitors that are selective inhibitors of the COX-2 enzyme. There are many drugs undergoing late-stage clinical trials at present for a number of human diseases ranging from cardiovascular disease to cancers that are based on information discovered by structural biology. It is expected that this approach to finding solutions to human health problems will accelerate in the future, as it is becoming increasingly important in the fight against newly-emerging and re-emerging viral and microbial diseases.

rational drug design is currently being applied to many areas of drug development. Anti-viral developments include efforts to abate the HIV pandemic; the serious human health risk posed by the hepatitis C virus, which is mutating a rate that makes vaccine treatment ineffective; and measles. which continues to kill over a million children in Africa alone. Currently, no drugs are available for many third world protozoan pathogens like sleeping sickness, and malaria is becoming increasingly resistant to current drug therapy, as are several microbial diseases like tuberculosis and staphylococcus aurelius infections.

Almost all drugs used in the treatment of cancer cause serious side effects because they lack selectivity for tumours over normal tissues. Selective activation relies on successful exploitation of the differences between the environment in tumours and that in healthy tissues. Tumour hypoxia, the lower than normal oxygen levels present in solid tumours, is the result of the rapid growth and vascularization of tumours. For a drug to be activated in an hypoxic environment, it must have an inactivated higher oxidation state and an activated lower oxidation state. To date, the development of hypoxia-selective agents has been carried out in the absence of information about the oxidation status of the agents in tumours and, in particular, how this status is affected by the degree of hypoxia. Extensive investigations of Co and Pt anti-cancer drugs using x-ray absorption spectroscopy are in progress to determine the oxidation state in situ in different regions of tumours, and in models of hypoxic tumours. Simultaneously, the project will provide information on the relationship between reduction potential and the extent of activation in hypoxic environments.

Vibrational spectroscopy and circular dichroism are complementary techniques that are able to monitor the take-up of anti-cancer drugs in cells and their effect on the conformational changes that these cause in critical proteins. Synchrotron light will add a new dimension to these studies because it will be possible to follow these processes in real time.

Cobalt-, copper-, nickel-, and zinc-based anti-inflammatory compounds are potent vetinary drugs and are likely to enter human clinical trials in the near future. X-ray absorption spectroscopy has been used extensively in the characterization of new drugs in the solid state, solution, pharmaceutical formulations and biological fluids. This research has been essential in determining the stability of the drugs in pharmaceutical preparations. X-ray absorption spectroscopy on the micron scale, which will be possible using the microfocus spectroscopy beamline at the Australian Synchrotron, is able to image the uptake and metabolism of metal-containing pharmaceutics in cells and tissues. This will provide a better understanding of the pharmacology of these drugs for the development of better and safer systems.