By Prof M W Parker
1 The century of biotechnology
The 20th century has been described as the century of biotechnology because of the development of powerful biological tools over the past few decades which in turn was underpinned by great fundamental advances in understanding biology at the molecular and cellular levels. Perhaps the greatest highlight was the completion of the Human Genome Project in which the genetic makeup of humans was deciphered. The genome was found to contain over 30,000 genes and a single mistake in certain genes can lead to serious hereditary diseases, many of which are fatal. Whereas genes are the “blueprint” of the body, the proteins they encode are the molecular engines of the body. The human genome project has revealed the chemical makeup of genes and proteins but the function of nearly two thirds of known human proteins remains a puzzle.
2 The importance of protein structure
The three-dimensional structures of proteins are essential for understanding protein function and activity. Detailed knowledge of protein structures has been vital for our understanding of numerous biological processes, from enzymatic reactions to immune evasion by viruses. The major technique for determining the atomic structures of proteins has been X-ray crystallography, although in the last decade nuclear magnetic resonance spectroscopy has proved a powerful tool for deciphering the atomic structures of small proteins (< 25,000 Daltons).
The discovery of X-ray crystallography and its application for solving structures of molecules was made by Sir William Bragg and his son, Sir Lawrence Bragg. Some of Sir William’s early work was performed in Australia when he was Professor of Natural History (Physics of the day) at Adelaide University. Sir Lawrence was born in Adelaide. Their achievements in X-ray crystallography were recognised with the award of the Nobel Prize in 1915. Protein crystallography can trace its origins back to 1934 when J.D. Bernal and Dorothy Crowfoot at the Cavendish Laboratory, Cambridge discovered that crystals of the stomach protease pepsin yielded an X-ray diffraction pattern (Bernal and Crowfoot, 1934). It was not until 1960 that the technical difficulties associated with deciphering an atomic structure from diffraction patterns of protein crystals were overcome and the first structure of a protein was published (Kendrew et al, 1960).
3 What is protein crystallography?
In order to visualise structures at the atomic scale it is necessary to work with electromagnetic radiation with wavelengths of the order of atomic bond distances (approximately 1 Å or 10 -10 metres). X-rays have such suitable wavelengths. X-ray diffraction – the interference between waves scattered from individual atoms – can be used to determine atomic structures. However, there are no lenses available to bend and focus the scattered X-rays. Instead atomic structures must be reconstructed using diffraction theory from the intensities of the diffracted waves which can be measured experimentally. Crystals, which are three-dimensional arrays of molecules, are required for X-ray diffraction experiments because scattering from individual molecules is far too weak to measure. Crystals act as amplifiers by increasing the scattering signal due to the multiple copies of molecules within them.
4 Structural genomics
One of the greatest scientific endeavours, the Human Genome Project, has recently been bought to fruition with an estimate that the human genome encodes about 30,000 different proteins. However, the functions of only about one third of these proteins are known with any certainty. Biologists are now embarking on the next big challenge: to decipher the function of all proteins in the human body. This new endeavour, coined by the term functional proteomics, is utilising a variety of powerful tools including X-ray crystallography. It has been argued by advocates that because the function of a protein is encoded by its three-dimensional structure, then structures will lead to a knowledge of protein function. At a practical level all new protein structures are compared to known structures deposited in the Protein Data Bank (http://www.rscb.org/ ), the depository for models of macromolecular structures. Similarities lead to hypotheses that can then be tested by biological assays. Because of the large number of protein structures that need to be determined from many different genome projects, there is a pressing need to speed up the process of solving structures from the current time of a few months to days. Such research, commonly referred to as structural genomics, is likely to lead to major technological improvements in the coming years.
5 Structure-based drug design
In the past, the majority of drug discoveries have been based on astute but serendipitous observations or by large screening programs of synthetic and natural products. Advances in molecular biology and protein crystallography have yielded a much more promising method termed rational or structure-based drug design (SBDD). Decades of research have demonstrated that proteins are the sites of action for most drugs and hence are the targets for the development of new drugs. In SBDD key proteins are identified, crystallised and their structures determined. Through the use of interactive computer graphics and molecular modeling software, it is possible to design potential drugs on the basis that good inhibitors must possess significant structural and chemical complementarity to their therapeutic target. The SBDD methodology requires an iterative procedure in which compounds are designed and synthesised, and crystal structures of the protein-drug complexes are then determined to test the modelling predictions. Successful examples of this approach include the currently used cocktail of HIV protease inhibitors against AIDS (Erickson et al, 1990) and neuraminidase inhibitors against influenza (Von Itzstein et al, 1993).
6 Why synchrotron light is so important to the study of protein structure
During the past ten years the number of protein structures elucidated by X-ray crystallography has risen more than tenfold. At last count there were more than 17,000 protein structures available in the Protein Data Bank. Driving this increase has been the use of synchrotron light sources to solve protein structures: from less than 30% in 1990 to more than 80% at the end of the last decade. There are over 50 synchrotron stations around the world dedicated to protein crystallography and nearly the same number are under construction or planned.
Synchrotron light provides two features essential to protein crystallography: speed and accuracy. The former is important in any structural genomics project and the latter is considered vital in structure-based drug design. Protein crystals are generally very small with typical dimensions of fractions of a millimetre. Quite often the diffraction data collected from a laboratory X-ray source are weak, and in some cases, not observable if the protein crystal is too small. It can take many months, and even years, to grow larger protein crystals. In some cases larger crystals cannot be grown at all. However, the high flux of X-rays from a synchrotron source can overcome crystal size problems in many cases. For example, the microfocus beamline at the European Synchrotron Research Facility in Grenoble, France has been used successfully to collect diffraction data off 5 micron crystals.
In the X-ray diffraction experiment the intensities or amplitudes of the X-rays scattered from crystals can be measured but the phase component of each wave relative to the others is lost. The central challenge in determining protein structures from diffraction patterns is to overcome the problem of determining phases for each measured diffraction spot. One way of determining phases is the use of so-called anomalous scattering that arises when the energy of the incident X-ray is close to the resonant frequency of the tightly bound inner shell electrons of an atom. Synchrotron light has revolutionised the use of anomalous scattering in solving protein structures because, unlike in-house X-ray sources, the wavelength of synchrotron light can be finely tuned near absorption edges to extract as much signal as possible.
7 Protein crystallography in Australia
Prior to 1990 there were only two protein crystallography laboratories in Australia: one in Sydney headed by Hans Freeman and another in Melbourne headed by Peter Colman. However, the last decade has seen an exponential growth in protein crystallography with now nearly 20 groups distributed around Australia. The success of all these groups has been partly dependent on the availability of synchrotron light sources, principally in Japan and the US. In recent years the Australian Synchrotron Research Program, funded as a Federal Government Major National Research Facility, has ensured good access for Australian protein crystallographers to the Advanced Photon Source in Chicago.
8 The future
In order for Australian protein crystallography groups to remain internationally competitive it has been clear that we must have access to our own synchrotron. The recent announcement that Australia will build its own synchrotron has been applauded by the Australian crystallographic community. We expect the synchrotron will provide new impetus for the further growth of the field in Australia and is likely to underpin the discovery of new pharmaceuticals by the structure-based drug design approach.
Bibliography
- Bernal, J. D. and Crowfoot, D. 1934. X-ray photographs of crystalline pepsin. Nature. 794: 133-134.
- Erickson, J., Neidhart, D. J., VanDrie, J. et al. 1990. Design, activity, and 2.8 Å crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science. 249: 527-533.
- Kendrew, J. C., Dickerson, R. E., Strandberg, B. E. et al. 1960. Structure of myoglobin. Nature. 185: 442-447.
- Von Itzstein, M., Wu, W-Y., Kok, G. B. et al. 1993. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature. 363: 418-423.
Glossary of protein crystallography terminology
- Proteins – the biological machines of the body. Many diseases are due to malfunctioning proteins.
- Protein crystallography – the study of the three-dimensional shapes of protein molecules by shining X-rays at protein crystals.
- Structural genomics – using the knowledge of the three-dimensional shapes of proteins to determine the function of proteins. This area of research will grow enormously over the next decade building on the recent successes of the Human Genome Project.
- Structure-based drug design - the computer-assisted design of drugs based on the three-dimensional shapes of proteins.
- Synchrotron light source – a machine the size of a football stadium for generating very intense electromagnetic light including X-rays.
- Human Genome Project – a major international project that has mapped the chemical make-up of all the genes in the human body.
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