Over the last few years, thanks to synchrotron radiation, we have witnessed a veritable revolution in molecular and structural biology. At present the seven beamlines of the ESRF dedicated to protein crystallography represent about a quarter of its scientific activity. Biologists are making more and more use of this tool to see, understand and control living matter better.
How far we have come since the discovery of the DNA double-helix, made with the assistance of X-rays half a century ago. We have now established the structure of thousands of proteins and, in some cases, protein crystallography has become a matter of routine. But scientists are now embarking on more and more difficult ventures, such as the study of membrane proteins or macromolecular complexes. Fortunately, synchrotron radiation, with its remarkable intensity, allows research to advance ever further. Among the challenges in the area of public health, the fight against viruses and bacteria requires structural information which is absolutely crucial in producing the medicines, vaccines and antibiotics of the future.
Alzheimer’s, malaria, avian flu...
To see the atomic structure of a protein using X-rays it is first necessary to make a protein crystal. It is only in this crystallised form that proteins will reveal their atomic structure under X-ray illumination. With synchrotron radiation, extremely powerful detecting instruments and very advanced computer software, highly complex protein architectures can be visualised and clarified.
Synchrotron crystallography is often an indispensable tool in the success of the most varied research projects. Because of the extremely narrow focus of certain rays it has been possible, for example, to identify the basic structure of the axis of amyloid fibrils, seen in Alzheimer’s, whose atomic structure is very difficult to observe.
The structure of the antigen AMA1 of the plasmodium which carries malaria, currently being tested as a vaccine candidate, has also been identified by crystallography at the ESRF, as has the structure of the hemagglutinin responsible for the virulence of the virus which caused the great Spanish influenza epidemic of 1918 (in which interest has recently been revived by new threats of a global avian flu epidemic), and that of recBCD, a protein complex which drives the repair of bacterial DNA.
Radiation resistance and DNA repair
Joanna Timmins, a member of the macromolecular crystallography team, is particularly interested in the bacterium Deinococcus radiodurans which has the fascinating property of resistance to very powerful doses of radiation. Its genome has been sequenced and some of its proteins crystallised. This bacterium has no exceptional characteristics, except its ability to repair any breaks in DNA double helices, which are generally fatal to cells, very effectively.
"Since this bacterium can resist levels of radiation not found on Earth, it cannot be an acquired characteristic. This resistance to radiation may be just an indirect consequence of its exceptional efficiency in a whole range of classic cellular functions – which would also explain, for example, its remarkable resistance to drought," says Joanna Timmins. If we had a better understanding of the fundamental mechanisms involved in repairing breaks in DNA in D. radiodurans it might help us to see how they occur in radiation-induced cancers, such as skin cancers.
In the same group, Laurent Terradot is working in collaboration with the Pasteur Institute on Helicobacter pylori, the bacterium responsible for gastric ulcers. Study of the interactions among the proteins involved in the detoxification of acidity – a process essential to the survival of bacteria in the stomach – has led to the discovery of a unique mechanism. "Helicobacter pylori accumulates nickel and then releases it when acidity increases. The released nickel allows another protein in the bacterium to trigger the process of detoxification. Last year the same team published the structure of the 12 proteins which comprise the machinery by which the bacterium injects its toxins into human cells. These are all proteins which could be targeted for therapeutic purposes".
A cure for gliomas?
In the field of radiology, methods developed by teams at Grenoble University Hospital in association with the ESRF have yielded highly encouraging results. They are based on a synergy between the treatment of tumours with radiation and a form of chemotherapy based on Cisplatine, a prominent antineoplastic used in attempts to treat gliomas – cerebral tumours for which at present there is no cure. "By selecting a specific platinum energy threshold for the radiation therapy, the absorption of cisplatine is multiplied. Laboratory rats will survive for a few weeks with radiotherapy or chemotherapy alone, a little longer when the two are combined. But with monochromatic synchrotron radiation at the right level, we see genuine remission being achieved – for the first time," explains Alberto Bravin.
Silk – source of bio-inspiration
Studying life is one thing – but copying it is an altogether different proposition. "Nature is an inexhaustible source of inspiration," says Christian Riekel, director of the Soft condensed matter team at the ESRF. "It was the resistance, elasticity and bio-compatibility of a thread of silk which gave us the idea of using it as a replacement for human tendons." These properties suggest many other uses too, as long as it proves possible to manufacture these fibres industrially.
The team is developing imaging techniques and conducting studies to understand better how this polymer of a very simple protein can form a filament in a water solution – just a routine task for the common spider. "Microfluidics, which uses chips containing tiny channels, or other comparable techniques, allows us to visualise at each moment, under the X-rays, how a protein in solution changes its configuration when one adds an aggregate product."
Other researchers are attempting to construct artificial muscles using a copolymer formed of a hard polymer and a soft polymer. "When the ambient acidity changes, this copolymer absorbs water, swells up and triggers a mechanical effect. This occurs on the level of a single fibril, microscopic in scale, but requiring a length of time that seems infinite when compared with the speed of natural muscle."
Silk, tendon or muscle – the interactions of the rigid protein fibres with the soft matrix surrounding them plays a major role in their mechanical properties. These fibres are immersed in a matrix to which they are anchored by links which rupture in the presence of water. What was, up to that point, a homogenous ensemble, then begins to break up. "How are these ruptures at the molecular level integrated and how do they transmit themselves to the entire muscle or tendon to change its state? How do these different organisational levels work together? To understand these processes we need to be able to study the same phenomenon at these different levels of organisation. Thanks to the nanometric precision provided by the ESRF beamlines we can now introduce techniques which allow us to study this transition from the microscopic to the macroscopic much more closely than was possible even ten years ago."
In synchrotron-radiation imaging, the rays can illuminate the whole sample or scan it point by point with a resolution of between 100 μm and 100 nm (0.1μm). This allows the presence and condition of trace elements like metals to be detected in the tiniest quantities. This type of detection...
Mary Rose, Stardust, arsenic and iodine
Analysis of the deterioration of the wreck of the Mary Rose, a ship belonging to Henry VIII of England. Courtesy of the Mary Rose Trust
In synchrotron-radiation imaging, the rays can illuminate the whole sample or scan it point by point with a resolution of between 100 μm and 100 nm (0.1μm). This allows the presence and condition of trace elements like metals to be detected in the tiniest quantities. This type of detection is used in a wide variety of research: archaeology, space research, environmental studies, new medical treatments, etc. Teams of scientists come to the ESRF, for example, to understand and prevent the formation of the sulphuric compounds which cause the deterioration of the timber in the wreck of the famous Mary Rose, the ship which belonged to Henry VIII of England. They also come to carry out chemical and structural analysis of the material brought back to Earth by NASA’s recent Stardust mission, to study the interactions between bone matter and a titanium prosthetic device, or to monitor the metabolisation of arsenic in a strand of hair.
Life through the eye of the synchrotron
