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Film Captures Proteins' Work

Photonics.com
Jun 2007
Kipp Lynch, PhD

GRENOBLE, France, June 20, 2007 -- If a picture is worth a thousand words, than a moving picture should be worth considerably more. While science may play a limited role in most Hollywood films -- except for the mad scientists who nearly destroy the world -- researchers at the Institut de Biologie Structural (IBS) and the European Synchrotron Radiation Facility (ESRF), both based in Grenoble, have given the enzyme superoxide reductase a starring role in their latest film.

Superoxide reductase, an enzyme found in some bacteria (a complex counterpart is found in humans), functions to eliminate the toxic free-radical species called the "superoxide radical." According to one of the principle researchers, Dominique Bourgeois of IBS, a detailed understanding of superoxide reductase is needed because the accumulation of superoxide radicals or other toxic forms of oxygen is involved in the aging process and in the development of disease.

proteinfilm.jpg


A film shows how the lysine amino acid (yellow part of the protein) grabs a water molecule (in blue) and imports it into the enzyme to perform the catalytic reaction on the superoxide (in red). (Image courtesy Gergely Katona)
"In humans, oxidative stress is associated with cancer or Alzheimer. Our work provides an important step in the understanding of how superoxide reductase actually works. The structure of the resting enzyme was already known, but here we captured the protein at work, en route to processing a superoxide molecule. The trick engineered by nature to make hydrogen peroxide (the product of the reaction) from superoxide could by large be pinpointed, although of course some questions remain," he said.

Traditional techniques such as x-ray crystallography only provide a direct view of enzymes and other biological macromolecules at near atomic resolution, but scientists cannot assume that what is seen in the crystalline state, where individual proteins are densely packed against each other, is the same in an organism, where the molecules are floating in the liquid cellular medium. Bourgeois and his colleagues applied in crystallo Raman spectroscopy to both phases of matter (crystalline and liquid) so that they could compare the two states and pinpoint eventual artifacts of the crystalline state.

"We found that Raman spectra from superoxide reductase were the same in the crystal and in solution, showing that the crystallographic x-ray structure we obtained was indeed biologically relevant," said Bourgeois.

In addition, whereas x-ray crystallography provides a global view of macromolecules at near-atomic resolution, Raman spectroscopy provides a local view with subatomic resolution. Some of the atoms, and the chemical bonds they form, can be observed precisely.

"The two techniques are perfectly complementary to each other. In the case of superoxide reductase, atoms that are essential to the catalytic mechanism of the enzyme (in particular an iron atom) are seen both by Raman and by crystallography," said Bourgeois. "Therefore, their chemical configuration can be established very reliably, allowing us to decipher how the enzyme functions."

The research team used the ESRF-IBS Cryobench laboratory to freeze the protein in three different states while the reaction took place. The in crystallo Raman spectroscopy gave them strong evidence that the states were the appropriate ones by showing them the chemical bonds in each stage of the reaction. Once they had identified the right states, they studied the sample with synchrotron x-rays.

In the last years, several groups of researchers made hypotheses as to how superoxide reductase functions, primarily based on spectroscopic or theoretical studies. The problem was that, in the absence of a direct view of the molecule in action, suggestions for a mechanism could only be indirectly inferred from the results.

"The binding geometry of the peroxide species was intensely debated. Most researchers thought it would bind in a so-called "end-on" fashion (that is only one of the two oxygen atoms would form a bond to the iron), but some others rather thought it would bind in a "side-on" fashion (in which case the two oxygen atoms bond to the iron)," said Bourgeois. "Our work provides evidence that the "end-on" binding mode does indeed occur. It does not exclude, though, that a "side-on" species may also form at a very early stage of the reaction. Therefore, our results will reconcile the community of researchers working on superoxide reductase."

ESRF has been filming proteins in action for several years, but until Bourgeois and his colleagues filmed the enzyme superoxide reductase, the experiments were limited to proteins that could be excited by light and are also very resistant in crystalline form.

"The achievement of this research is twofold: On one hand there is the technological success of filming an enzyme in action and on the other hand there are the results that contribute to the knowledge of how this enzyme works," said Bourgeois. "The catalytic mechanism of an important enzyme involved in oxidative stress has been unveiled. This was done thanks to an original combination of crystallography and spectroscopy, which will be applicable to the study of many other biological systems."

For more information, visit: www.esrf.eu


GLOSSARY
photonics
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
raman spectroscopy
That branch of spectroscopy concerned with Raman spectra and used to provide a means of studying pure rotational, pure vibrational and rotation-vibration energy changes in the ground level of molecules. Raman spectroscopy is dependent on the collision of incident light quanta with the molecule, inducing the molecule to undergo the change.  
resolution
1. In optics, the ability of a lens system to reproduce the points, lines and surfaces in an object as separate entities in the image. 2. The minimum adjustment increment effectively achievable by a positioning mechanism. 3. In image processing, the accuracy with which brightness, spatial parameters and frame rate are divided into discrete levels.
synchrotron
A device that uses superconducting magnets to bend or accelerate charged particles. It can be used to etch very fine high-density patterns on integrated circuits.
x-ray crystallography
The study of the arrangement of atoms in a crystal by means of x-rays.
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