Melbourne researchers have used the Australian Synchrotron to produce the first three-dimensional structure of a molecular scaffold, known to play a critical role in the development and spread of aggressive breast, colon and pancreatic cancer.

Armed with the structure, the research team is looking at ways of targeting parts of the scaffold molecule critical for its function. They hope the research will lead to novel strategies to target cancer.

The research was the result of a long-standing collaboration between Walter and Eliza Hall Institute (WEHI) researchers Dr Onisha Patel and Dr Isabelle Lucet and Monash University Biomedical Research Institute researcher Professor Roger Daly.

Dr Santosh Panjikar, a macromolecular crystallographer at the Australian Synchrotron and Dr Michael Griffin from Bio21 Institute at the University of Melbourne made important contributions to the study, which was published in the journal Nature Communications.

Lucet said SgK223 is a member of a family of proteins called pseudokinases and had been classified for a long time as a ‘dead enzyme’. 

“SgK223 doesn’t have the measurable activity that we see with other types of enzymes, and this meant it was largely ignored. However in the past decade, we’ve come to understand that this ‘dead enzyme’ plays an active and important role in cell signalling,” Lucet said.

MX2 beamline used to determine crystal structure 

Panjikar explained that measurements on the macromolecular crystallography beamline, MX2 and small angle X-ray scattering on the SAXS/WAXS beamline were used to determine the crystal structure of SgK223 and oligomeric state of SgK223 in solution respectively.

“There were many challenges working on this protein,” said Panjikar.

Initial experiments on crystals of SgK223 were not successful because the protein is highly sensitive to various heavy atom solutions, and to X-ray radiation and deteriorates very quickly. 

“Protein crystals are normally sensitive to radiation but the selenomethionine protein crystals were more sensitive,” said Panjikar.

 “You want to collect your data in a way that doesn’t damage the crystal but retains the anomalous signal,” said Panjikar. 

"We designed a diffraction strategy for the sample, in which we used a small sized beam 20 microns across and clad at several places going from one position to the next on the rod-shaped crystals.”

The researchers collected multiple data sets at different X-ray energies around selenium edge for multiple wavelength anomalous dispersion (MAD), a specialised technique that allows them to use ‘tunable energy’.

“Selenium has an absorption edge at particular energy where it absorbs more X-rays. We went on to collect X-ray data from Sgk223 crystal at the higher energy side of that edge and also at below the edge,” explained Panjikar.

They were able to solve the crystal structure using a software pipeline, Auto Rickshaw, developed by Panjikar.

“Where you have multiple data sets at different energies, you need to check which data set will actually work. In this case, with Auto Rickshaw we found one combination of the data sets that worked very well,” said Panjikar.

The data was used to get the preliminary phases and electron density map, which enabled the researchers to build the 3D model.

“When you determine the crystal structure of the protein you know what the molecule looks like but it also confirms if the molecule is a monomer or dimer,” said Panjikar.

SAX confirms crystal structure

“We could see that SgK223 was a dimer, but needed supporting confirmation that what we saw in the crystal structure was the same in solution. “

Validation of the dimer was achieved using small angle X-ray scattering of the native protein.

Other biochemical techniques carried out at WEHI, Monash and the University of Melbourne were used in the study.

“The world-class facilities at the Australian Synchrotron in Melbourne were instrumental in the discovery,” co-author Lucet said.

“It is the only facility in the Southern Hemisphere that has the specialised technology required to provide us with detailed knowledge essential for seeing molecules at an atomic level. This is essential if we wish to discover and develop drugs that target and interfere with molecules that drive cancer and other diseases.”

Media release 

Read more about the research on the Walter and Eliza Hall Institute of Medical Research.

DOI: 10.1038/s41467-017-01279-9

 

Prof Andrew Peele, Dr Richard Garrett and Australian Consul General at the Osaka Consulate David Larson at the 20th anniversary event

Prof Andrew Peele, Director of Australian Synchrotron (above left) and Dr Richard Garrett, Senior Advisor Synchrotron Science, Strategic Projects, Industry and External Engagement (above centre) attended the 20th anniversary of user operations at the SPRing-8 synchrotron, in Hyōgo Prefecture, Japan last week.

 

They were among 500 guests, industry leaders and politicians from Japan, and directors of the majority of the world's major synchrotrons, who gathered at the historic Himeji Castle also in Hyōgo Prefecture to mark the occasion.  

 

There were photographs on display of all the synchrotrons that sent directors or representatives to the event.

 

“It was an opportunity for those of us who manage synchrotron facilities to congratulate SPRing-8 on the milestone and get together to discuss the application of synchrotron science to meet future challenges,” said Prof Peele.

 

“Synchrotron science using X-rays and infrared light continues to be a powerful and invaluable tool in investigating the nature of materials for practical applications.” 

 

The SPRing-8 synchrotron, the world’s largest 3rd generation synchrotron opened to national and international users from industry, academia and government in 1997.  The synchrotron radiation facility operates with a beam energy of 8 GeV with 62 beamlines.

 

Third-generation synchrotron radiation facilities are designed especially for installing as many insertion devices as possible in a dedicated storage ring.

 

The anniversary ceremony was followed by a symposium on "Synchrotron Radiation for the Future of Humanity", which held in the 17th century castle.  

 

The Australian Consul General at the Osaka Consulate, David Lawson (above right) , also attended. 

 

ANSTO has an international partnership with the SPRing-8 synchrotron.

 

 

 

A large international collaboration has used a specialised technique on the infrared microspectroscopy (IRM) beamline at the Australian Synchrotron to determine the structure of proteins in individual silk fibres that has potential use in the design of new biomaterials with desirable properties.

The technique, hyper-spectral infrared imaging, is a powerful analytical tool because it can establish the link between micro-/nano-structures and specific material properties of biomaterials. 

The orientation of the C = O, C-N, and N-H bonds in amide structure of the L-section of silk fiber confirmed in this study by the hyper-spectral imaging

The investigation included researchers from Swinburne University, Tokyo Institute of Technology, Deakin University, the Australian Nanofabrication Facility, The Centre for Physical Sciences and Technology in Lithuania, Dr Mark Tobin and Dr Pimm Vongsvivut from the Australian Synchrotron, in a study that was published in Scientific Reports

The extraordinary properties of silk are linked to the molecular orientation of polypeptides and its amorphous/crystalline composition in the protein structure. 

“The goal was to identify the orientation of proteins in different parts of the fibre and to look at how laser treatment can alter the protein structure in the silk fibre,” said Dr Mark Tobin, Principal Scientist ‒ IR beamline at the Australian Synchrotron. 

“You would need to know the effect of a laser on silk, for example, in order to 3D print the silk,” said Tobin.

Molecular orientation is responsible for the optical, mechanical and thermal properties of biomaterials. In this study, the researchers were interested in investigating the molecular orientation of specific protein bonds in the silk that play a critical role in its strength.

Infrared imaging at the Australian Synchrotron can access molecular orientation of the protein structure directly from a single silk fibre.

“You can obtain infrared absorption information that is selected based on the orientation of a particular chemical bond,” explained Tobin.

Hyper-spectral imaging

“Because the silk fibres are only 10 microns across and the synchrotron infrared beam is about half the size of that, we developed an optical device using a germanium crystal that allowed the beam to pass through the fibre’s cross section at four times higher resolution.”

This specific device, which was developed by Vongsvivut and Tobin at the Australian Synchrotron, was recently used successfully on carbon fibres and has shown to be efficiently suitable in a broad range of applications.

Silk is a semi-crystalline material that is birefringent, which means as well as absorbing polarised light in one way it actually rotates the polarisation.

The researchers used an infrared filter to progressively rotate the polarisation of the synchrotron beam and collected four infrared (chemical) images ‒ each one with the polarisation 45 degrees apart. This unique four polarisation method was developed by the collaborative researchers in Japan. Using a mathematical formula to transform the polarisation data, they were able to work out the molecular orientation of the protein structure in the silk fibres.

Infrared imaging

In an infrared image, the intensity of the colour indicates the strength of the absorbance. 

“In the infrared wavelengths, you see peaks in the spectra that tell you where the light is being strongly absorbed,“ said Tobin.

“A bond vibrates at a certain energy level at a natural frequency. If light comes in at the same frequency, it can absorb some of that infrared light and vibrate to a slightly higher level,” explained Tobin.

High resolution 1.9 μm ATR FT-IR maps at 1.9 μm resolution of the longitudinal (L) cross sections of silk presented in auto-scale for better viewing

The spectra generated in infrared images revealed that the primary vibration of the Amide II bond was all along the direction of the chain and the vibration of the Amide A bond was perpendicular to the fibre.

“With that information, our collaborators were able to work out that the protein molecules oriented in a particular way in the fibre.”

When a pulsed laser was used on one of the bonds, it disrupted the Amide A bond, changing the protein structure.  

“Although the bulk information of silk fibres has probably been known, it has not been possible to measure molecular orientation on single fibres before,” said Tobin.

doi:10.1038/s41598-017-07502-3