Shining a light on membrane proteins

Membrane proteins are pretty awful molecules to work with. Unlike soluble proteins that often happily crystallise, the hydrophobic surfaces of membrane proteins make crystallisation a nightmare, and yet they are such important proteins for signal transduction and molecular transport that structural biologists are very keen to have a good look at them.

Only a handful of membrane protein structures have been elucidated, mainly using cryoelectron microscopy; the rest remain in the great unknown. While some researchers continue to refine X-ray crystallography techniques and electron microscopy to hunt down and uncover these recalcitrant molecules, others are looking at different approaches to the problem.

Possibly the most promising approach is to use coherent X-ray diffraction from single molecules. This technique is similar to X-ray crystallography in that a diffraction pattern is measured, however it has a major benefit - there is no need to crystallise the proteins. Instead, a coherent beam of x-rays - a targeted beam, like a laser - is shone at a single object and its diffraction pattern is measured.

At the ARC Centre of Excellence for Coherent X-ray Science (CXS), researchers have already used the technique to obtain images of cells and are working their way towards individual molecules. For example, biochemist Professor Leann Tilley from La Trobe University, who is also Deputy Director of CXS, has tested the technique on malaria parasite-infected red blood cells and come up with the goods.

"With higher and higher intensity sources, you may be able to get the structures of individual proteins just from their individual diffraction patterns," Tilley says. "The holy grail is to get molecular information about cell components without taking them out of that cellular environment. That is the promise of x-rays."

Tilley's colleague at La Trobe and leader of the Biological Sciences Program at CXS, Associate Professor Mike Ryan, is working on mitochondrial diseases and is particularly interested in some of the membrane proteins involved in disease processes. He too is interested in whether coherent diffraction imaging can give him structural information about essential membrane proteins without having to crystallise them.

---PB--- Phase imaging

The idea behind the centre came from physicist Professor Keith Nugent, best known for his work in phase imaging, a way of measuring light refraction from materials in a quantitative manner.

Phase imaging has been used quite extensively in materials science and for physical structures and is now being applied to biological structures.

"If you look at a cell without staining, you basically can't see anything," Nugent says. "But the cell is affecting the light, the phase of the light. It's just like a lens - they are transparent but they affect the light by bending it. Phase imaging is rendering that aspect of the light visible."

Techniques Nugent has helped develop such as X-ray phase contrast has been used to create the beautiful images we can now see of tiny insects fossilised in amber. The ideas are now being applied to structures within living cells.

"With protein crystallography, you shine X-rays at the crystals and you get the diffraction pattern from an array of proteins; from that you can determine the molecular structure," he says.

"Coherent X-ray diffraction applies those same ideas except you don't need crystals. You shine a coherent beam of X-rays onto a single object and measure the scattering of the X-rays. You don't need a focusing lens for this so you are not limited by the resolution of the lens. By inverting the diffraction pattern you can generate very high resolution images."

The CXS Biological Science group has long worked with both light microscopy and electron microscopy but sees X-rays as a new way forward. Leann Tilley is studying Plasmodium falciparum in red blood cells and the way the parasite manages to avoid being detected. The parasite produces proteins that make the host cell membrane stick to receptors on capillary walls.

"The parasite lives inside a vacuole in the cell and it needs to get the adhesion proteins out to the red cell membrane" she says.

"The parasite subverts the red blood cell's physiology and converts it from a one-function a sack of haemoglobin to a more fully functional erythrocytic cell. It puts membrane structures into the red cell's cytoplasm, which it uses to export the adherence proteins and insert them into the red cell membrane. It is these structures which we would like to image at very high resolution."

Tilley's team currently uses light microscopy imaging using green fluorescent protein, which is useful for labeling specific proteins and watching what is happening, but light microscopy is limited by its low resolution, she says.

"Electron microscopy also works really well because you get really good resolution, but because electrons can't get through matter very readily you have to work with really thin slices. X-rays are in between. The wavelength of X-rays is in between the wavelength of electrons and the wavelength of light, so you get increased resolution, but X-rays also have very good penetrating powers so you can see the inside of the cell, rather than just the surface."

---PB--- Light in femtoseconds

Keith Nugent, a Federation Fellow, developed the idea of a collaborative centre in order to build up capacity in X-ray imaging in Australia.

Nugent has worked with biologists on developing microscopy techniques for many years, and has also worked in synchrotron-based phase contrast imaging. He is keen to harness the potential of the Australian Synchrotron, which is conveniently close.

"One thing we are doing in the centre is developing an imaging end-station which we'd ultimately like to set up at the Australian Synchrotron," he says.

"We currently don't have the necessary infrastructure in Australia and we will initially put it at the Advanced Photon Source in Chicago. However we are putting together a collaboration with universities around Australia to raise the money to bring the facility back to the Australian Synchrotron in a couple of years time."

Raising funds for a dedicated beamline at the Australian Synchrotron is also in the plan. CXS researchers already use the Australian Synchrotron, says the centre's chief operating officer, Tania Smith.

"We already use it to do some experiments but the Australian facility does not yet have all the facilities we need." Smith says. "We also have a memorandum of understanding with the CRC for Biomedical Imaging Development and we'll work with them on a project to build X-ray detectors for biological samples."

CXS works as a series of nodes, including the University of Melbourne, La Trobe University, Monash University and Swinburne University of Technology. CXS also has a major partnership with scientists in CSIRO, an activity that is treated as a CXS node, though it is funded by CSIRO. Each node specialises in different areas and the centre has access to a great array of equipment, including a new Femtosecond High-powered Laser Facility at Swinburne, about which Nugent is rather excited.

"It's a very high powered laser beam that produces pulses with durations of tens of femtoseconds," he says. "If you fire these pulses into a gas, coherent X-rays come out the other side; which is quite miraculous."

The centre is half-way through its five-year funding period but is planning to continue beyond this. At the end of that, the centre aims to have developed a novel imaging technique that will provide information on the structures of membrane proteins.

"We would like to have developed a flexible form of very high resolution microscopy," Nugent says. "We want to do imaging of cellular samples in three dimensions down to a resolution of 10 nanometers, which is 10 to 20 times better than we can achieve with the best confocal microscope. And also we'd like to have made a major contribution to the conceptual foundations of single molecule imaging."