Heart of the scatter

Finding ways to 'see' the structure of molecules and viruses isn't easy. Now a revolutionary technique that took decades of work in the physical sciences to develop could help scientists probe right to the heart of biomolecules.

More than thirty years ago a small difference in the way handed (or 'chiral') objects scatter left and right-handed beams of light was discovered. This discovery led Laurence Barron and colleagues to develop the powerful analytical technique of Raman optical activity (ROA) spectroscopy. The technique is helping researchers study the structure of everything from small chiral molecules to intact viruses.

As a young researcher at Oxford University working with Peter Atkins, Laurence Barron began theorising about light interference and what happens when chiral molecules scatter left- and right-circularly polarised light.

"The fundamental mechanism responsible for Raman optical activity (ROA) was discovered as part of my DPhil work with Peter Atkins," Professor Barron told Newsline. Barron moved to Cambridge University in 1969 to start postdoctoral work with David Buckingham in a purportedly different area, supported by a Postdoctoral Fellowship from the Science Research Council. "I found that he was thinking along similar lines," says Barron, "so we joined forces to develop a more definitive and accessible theory of ROA."

Together with Martin Bogaard, an experi-mental Research Assistant of David Buckingham, the team set about trying to make the first observations of ROA. They obtained funding from the National Research Development Corporation in exchange for long-expired patent rights, but the work was hard. "It was a very difficult and marginal experiment," Professor Barron recalls, "given the technology available at the time, but we were eventually successful."

The researchers began experimenting with typical small organic molecules as neat liquids and published their findings in the Journal of the American Chemical Society in 1973. "This was an exciting discovery," comments Professor Barron, "as well as ROA being a completely new phenomenon based on a newly discovered fundamental mechanism, it was the first observation of vibrational optical activity in typical chiral molecules in the liquid phase."

Conventional optical activity phenomena such as optical rotation and circular dichroism of visible and ultraviolet light had been known about since the Nineteenth Century, but this new finding was eventually to lead to a breakthrough in spectroscopy.

Backscattering breakthrough

Barron took his growing expertise to Glasgow as a lecturer in 1975, where for the next fifteen years he continued to build on both the fundamental theory of ROA and also on experimental measurement.

The experimental work was sustained by grants from the SRC and subsequently the SERC. Working with post-doctoral researcher Lutz Hecht in Glasgow and Werner Hug in Freiburg, Switzerland, during the late 1980s ROA was ultimately measured in backscattered light rather than in perpendicular light, which is much easier. This result would prove crucial in subsequent applications in the life sciences. The research was also advanced by emerging technologies like charge-coupled devices (CCDs), highly efficient edge filters, and single-grating spectrographs.

"Out of the blue, I obtained a grant of £100,000 pounds from the Wolfson Foundation, which enabled us to build a completely new generation of instrument based on backscattering plus this new technology," Professor Barron explains. "This Wolfson-funded instrument propelled us into ROA studies of biomolecules in aqueous solution."

The biological connection means that since the mid-1990s, the ROA work in Glasgow by Barron, who currently holds the Gardiner Chair of Chemistry, and Hecht, currently a Reader, has been well-supported by the joint Bi-molecular Sciences Committee of BBSRC/EPSRC, and by the EPSRC itself.

The team has studied countless biomolecules from peptides and proteins to carbohydrates and nucleic acids, and even viruses. "There have been several other ROA instruments in other countries, but none has had the sensitivity to measure ROA spectra of proteins and viruses," says Professor Barron, "Our results show that ROA provides a completely fresh perspective on aqueous solution structure and the behaviour of biomolecules, which makes it invaluable for studying problems at the forefront of bi-molecular and biomedical science, such as protein structure, protein misfolding and disease, and the molecular structure of viruses."

The concept of protein misfolding is inherent in devastating diseases such as Alzheimer's and Creutzfeldt-Jakob disease (CJD), where misshapen protein structures damage brain cells irreparably.

If ROA spectroscopy can reveal how normal folding and abnormal misfolding take place, researchers might be able to better understand why these diseases occur and how to treat them. "I don't have any particular specialisation in studying biomolecules," confesses Professor Barron, "I have applied ROA to anything I laid my hands on." ROA has, he adds, been particularly useful in studying protein states associated with misfolding and disease.

This unique and pioneering work has stimulated Werner Hug to build an ROA instrument in Switzerland of completely new design, faster and easier to use than the current instruments being used by Barron and colleagues. US company BioTools Inc is now delivering the first production models of this instrument.

The same ROA instrument is important not just for studying biomolecules. "It can provide spectra of the whole range of chiral molecular structures, from the smallest such as bromo-chlorofluoromethane (CHFClBr) to the largest such as intact viruses," adds Barron. The small chiral molecule CHFClBr is of fundamental interest to chemists and Barron's team was first to determine its absolute configuration using ROA in 1997.

Probing proteins

There remains, however, a dearth of infor-mation on several key aspects of polypeptide research to which ROA spectroscopy may provide new insights. "Since ROA is a more incisive probe of peptide backbone conformation than other types of spectroscopy, it provides more information about the three-dimensional structures of proteins in aqueous solution," explains Barron.

One recent EPSRC-funded project for studying polypeptides and model conformations has provided definitive ROA signatures of these key elements of proteins. "The results have proved of immediate value in interpreting ROA spectra of proteins," says Barron. ROA can now distinguish between the common but irregular sheet elements found in most proteins and the extended flat sheets that are thought to be involved in prion diseases.

"The most amazing results to date are on intact viruses," enthuses Barron, "which give good ROA spectra from which information about both the coat protein structure, the nucleic acid core structure, and protein-nucleic acid interactions may be deduced." There are thousands of viruses about which little molecular structural information is known. ROA could make that valuable information accessible. "ROA gets right into the chiral guts of a biomolecule," adds Barron, "it reaches those parts others fail to reach, it's the 'Heineken' of spectroscopies!"