Feature: Australian Synchrotron transforms proteomics

Melbourne X-ray crystallographer, Tom Caradoc-Davies, needed only one hour on the Australian Synchrotron’s beamline to obtain the high-resolution diffraction data that revealed the structure of a key component of bacterial pili.

With old technology – an ‘in-house’ or ‘home’ X-ray source, in the jargon of the profession – it would have taken years to grow crystals of the protein to sufficient size for study, weeks to collect the raw data and a year to analyse it. Caradoc-Davies says he could have spent 20 years – half his research career – on the project.

Caradoc-Davies, who is principal scientist of the macromolecular crystallography beamline team at the Australian Synchrotron, says the brilliance of the synchrotron beam, and the capacity to tune its energy to the properties of the target macromolecule, yield sharp, high-resolution X-ray diffraction of a huge variety of macromolecules. It is delivering enormous time and cost savings to Australia’s biomedical research community, and accelerating research advances.

“In one second, I can obtain a diffraction image that would have taken up to an hour to produce with a home X-ray source,” Caradoc-Davies says. “In place of half a dozen fuzzy spots on an image, you now see a complete diffraction pattern.”

He says another big advantage of the synchrotron is that the brilliant, narrow beam means researchers to work with much tinier protein crystals than in the past, when crystallography with diffuse, low-powered X-ray sources demanded large, perfect crystals of proteins that were sometimes extremely reluctant to crystallise.

These capabilities make the Australian Synchrotron tremendously popular, says Caradoc-Davies. So popular, in fact, that the beamlines are heavily oversubscribed, with users applying for beamtime months in advance.

---PB---

Driving proteomics

Since the completion of the Human Genome Project in 2003, biomedical research has been increasingly driven by proteomics – the study of the structure and function of the myriad proteins coded by the near-biblical flood of animal, plant, bacterial and viral genes cascading into international gene data banks.

Without the synchrotron’s speed and precision, researchers might have taken a century or more just to work through the human genome’s catalogue of prospective targets for new therapeutic drugs and antibodies for infectious, inherited and metabolic diseases.

The front-line influenza drug, Relenza, is a showcase in the power of X-ray crystallography in the development of new therapeutics. Relenza was conceived in Australia in the 1980s, emerging from four years of painstaking work by CSIRO X-ray crystallograpers, Peter Colman and Jose Varghese, to solve the structure of the influenza virus’ neuraminidase coat protein. Colman and Varghese identified a pocket-like structure in the protein that is invariant in all forms of the highly mutable virus, including the current H1N1 swine flu pandemic strain, and the deadly H5N1 bird flu. Relenza blocks the pocket, trapping newly formed virus particles in infected host cells, and disrupting the virus’ replication cycle.

Relenza was the prototype for a new generation of ‘designer’ anti-viral drugs. Next-generation insecticides, fungicides, herbicides and biological control agents based on genetic manipulation will depend increasingly on rapid advances in proteomics, made possible by the synchrotron’s piercing, collimated X-ray beams.

Solving a protein’s structure provides clues to its function, and the 20,000-odd genes in the human genome code for as at least 10 times that number of proteins. They include cell-building structural proteins, enzymes – the genome’s high-energy ‘verbs’ – a slew of signalling proteins and their multiplicity of receptors, and transcription factors, which orchestrate the coordinated activity of dozens to hundreds of proteins.

Caradoc-Davies says the ability of the Australian Synchrotron to tune the beam’s energy to the sample makes it possible to extract extra structural information, by solving the so-called phase problem in X-ray crystallography. The energy of a ‘home’ X-ray source cannot be tuned, so phase information is lost.

“Phase information is a key part of the data needed to solve a crystal’s structure,” he says. “By substituting atoms of a metal like platinum, gold, mercury or selenium at known positions in a protein’s structure, you can collect data at an energy where the metal atoms produce anomalous scattering.”

This anomalous scattering creates ‘patterns’ in the X-ray data and the differences between the scattering pattern of the ‘doped’ and native proteins reveals where the metal atoms reside in the folded, crystalline protein. The metal atoms provide reference points for generating phases that reveal details of the structure of the native protein.

---PB---

Paper proliferation

Caradoc-Davies says the Australian Synchrotron’s advanced capabilities have allowed Australian and New Zealand researchers to produce a stream of high-impact papers since it was commissioned.

“Our work falls into three basic areas,” he says. “One is to develop a fundamental understanding of what is going on in biological systems, another is the study of specific disease-related problems, and the third is drug design and development.”

Important recent publications include a Cell paper by Dr Jacqui Gulbus and Oliver Clarke, of the Walter and Eliza Hall Medical Research Institute (WEHI), describing how gated potassium channels on the surface of neurons and other cells – most notably, heart muscle – open and close in response to regulatory signals.

The study revealed that the potassium channel contains an intracellular domain that acts as a switch for a filter domain that selectively allows potassium ions – but not sodium ions – to flow across the membrane.

Dr Sheena McGowan’s team at Monash Medical Research Institute published a paper in Proceedings of the National Academy of Science describing the structure of the M17 aminopeptidase in the malaria parasite, Plasmodium falciparum, a potential target for new anti-malarial drugs.

M17 is one of two neutral aminopeptidases that digests proteins in the host red blood cell to provide an intracellular pool of amino acids on which the parasite feeds. A ‘designer’ M17 blocker would starve the parasite, disrupting its replication.

McGowan and her colleagues used the synchrotron to solve the structure of M17, alone and complexed with two candidate inhibitors that target a hydrophobic pocket with an active metal-catalysed core that explains its avidity for certain host-cell proteins.

Monash University researcher, Dr Jamie Rossjohn, has published a number of papers in leading journals on the way Major Histocompatability Complex Class 2 molecules on cytoxic T-cells recognise certain peptide sequences in viral antigens displayed by infected cells (see Australian Life Scientist, July/August 2009, page 36). The findings should lead to improved vaccination strategies.

Caradoc-Davies says the synchrotron allows researchers to obtain ‘snapshots’ of signalling proteins or enzymes as they change conformation upon contacting receptors or substrate molecules. While the blindingly fast reactions cannot be tracked in real time, the interactions can be chemically ‘frozen’ at different stages and provide a sequence of structures revealing the protein’s dynamic behaviour.

“We can also monitor atomic vibrations of the atoms in the crystal structure, which provides information about the movement of the structural units crucial to the protein’s function,” he says. “All kinds of researchers work here – we have people working on Mycobacterium infections, like tuberculosis, and other infectious agents, like Staphylococcus and Streptococcus.

“Others are working on malaria and different cancers,” Caradoc-Davies says. “The cancer work falls into two streams. One is pro-apoptotic self-surveillance in cells, and what goes wrong when cells become cancerous, which involves solving the structures that induce natural killer cells to attack and destroy mutant proteins.

“That includes investigating anti-apoptotic proteins like Bcl-2 and Bax, and determining the structure of anti-apoptotic viral proteins that mimic their structure. The other program is exploring the signalling systems that induce natural killer cells to attack cancerous or virus-infected cells.

“Both of our protein structure beamlines are fully occupied. Just two lines are producing about 60 per cent of the synchrotron’s published output. Before the synchrotron was built, researchers used to have to go overseas to use synchrotrons in Japan and the US. In those days we would have been advancing at only a few percent of the pace we are today.”

---PB---

Beaming out cancer

About 50 per cent of cancer patients undergo therapy with ionising radiation to treat their malignancies. Professor Peter Rogers, of Monash University, says patients typically require a total absorbed dose of 50 to 70 grays in 2-Gy doses, which can involve up 25 to 35 hospital visits over six to 11 weeks.

Conventional radiotherapy uses divergent high-energy, low-dose beams, often with multiple beams angled to intersect within the tumour volume and deliver a higher dose to cancerous cells, while minimising damage to healthy tissues in their path.

Three years ago, Rogers began investigating a promising but, in his own words, “absolutely experimental” new radiotherapy technique, known as spatially fractionated microbeam radiation therapy (MRT).

MRT requires a third-generation synchrotron, and Rogers was using a $500,000 venture grant from the Victorian Cancer Council to commute to Japan to use the medical beamline at the Spring-8 synchrotron in Hyogo Prefecture. He is now working at Australia’s own third-generation synchrotron, backed by a $600,000, three-year NMRC Project Grant.

“A synchrotron generates a very high photon flux, and after passing through a collimator, the beam’s rays are approximately parallel,” Rogers says. “The primary beam is passed through a collimator, and emerges spatially fractionated into an array of microbeams, each 25 microns wide, and spaced at 200-micron intervals.

“Within each microbeam, the radiation level is hundreds of grays. The high intensity radiation is concentrated in the path of the microbeams, but scattering also directs some radiation into the ‘valleys’ between the beams.

“The appeal of MRT is that the beam is spatially fractionated, where conventional radiotherapy is temporally fractionated. Like conventional radiotherapy, MRT can also be delivered in intersecting beams. It has a significant impact on tumours, while causing significantly less damage to healthy tissues.”

A problem with MRT is that a synchrotron is immovable, so the patient has to be oriented in the beam’s path. “I’ve been to a few other synchrotrons to look at solutions, and I was very impressed with the system developed for the synchrotron in Saskatoon, in Canada. It’s a chair with three axes of rotation, which allows the patient to be oriented very accurately to the beam.”

Rogers says while it may be possible to create a dedicated radiotherapy facility at a synchrotron, it would accommodate only a limited number of cancer patients. “Like many technological advances, if it’s going to have a significant impact, it will be by stimulating the development of equivalent, compact photon beam sources that will be dedicated just to radiotherapy.

“We’re very much at the animal model stage, and the Australian Synchrotron’s medical beamline is still being built. Last year, we received $14.3 million in NHMRC funding to build a facility that we envisage will ultimately be used to treat human patients with MRT, but it’s still only in the planning stages.”