Singlet oxygen: from damage to proteins to the development of biosensors and generators – SOxygen

We chose to combine three different biophysical methods, since the effect of singlet oxygen on proteins is heterogeneous. We will use a high-resolution structural method, in combination with mass spectrometry which is capable of detecting small protein mass variations, and optical spectroscopy, which will allow us to quantity the extent of damage for certain samples.

All information obtained in this project should give us insights on two complementary aspects, one of fundamental research, one of biotechnological interest. We will be able to provide new ideas on how to evolve proteins capable of sensing or generating singlet oxygen, while collecting an outline of singlet oxygen effects on a variety of proteins. Our goal is thus to tame singlet oxygen in order to exploit its beneficial uses.

Non-applicable after only 6 months (see previous paragraph)

We have just published an article in Acta Crystallographica D: Biological Crystallography on a structural study of a fluorescent protein, which shows that its chromophore is well less protected from the solvent than we thought. Indeed, when cryoprotecting protein crystals with the small molecule ethylene glycol, we have observed that it was able to penetrate quickly within the protein, and binds next to the fluorescent chromophore. This result has implications on oxygen photosensitization into ROS (Reactive Oxygen Species) such as singlet oxygen.
‘Alteration of fluorescent protein spectroscopic properties upon cryoprotection’ by David von Stetten, Gaëlle O. Batot, Marjolaine Noirclerc-Savoye and Antoine Royant, Acta Crystallogr. D, in press.

Singlet Oxygen (SO) is, in physiological conditions, one of the most elusive reactive oxygen species (ROS). Although its physics and chemistry have been intensively studied over the last three quarters of century, providing it with a defining role in chemical synthesis, several open questions remain regarding its involvement in biological processes: is SO implicated in the oxidative burst of mammalian phagocytes in addition to superoxide and hydrogen peroxide? How does SO contribute to general aging when altering proteins? How does SO signaling work in plants?

Besides its physiological role, SO has become a topic of research for the development of biotechnological tools. Because of the non-specific inactivation properties of SO, a genetically encoded fusion protein with the ability of generating significant amounts of SO has long been desired. Such a protein would immediately find two applications. The CALI technique (for Chromophore-Assisted Light Inactivation) relies on the inactivation of a SO-generator-tagged protein of interest via light irradiation. The second application would be its use in high-resolution protein colocalization by correlated fluorescence and electron microscopy. Finally, developing a genetically encoded sensor specific for SO would be extremely useful in the study of the physiological role of SO in living cells.

At the University of California San Diego, Prof. Roger Tsien, recipient of the 2008 Nobel Prize in Chemistry, has long been a leader in the development of such tools over the last decade or so. Some of them have not been published yet, and are extremely promising. The coordinator of the project, Antoine Royant, has been a scientific visitor in the Tsien lab, and has become familiar with these tools. He is convinced that structural biology can have a definitive impact on the optimization of these tools. At the Institut de Biologie Structurale in Grenoble, we possess world-class competence in the combination of X-ray crystallography and optical spectroscopy to gain deeper understanding of the mechanism of proteins. This is materialized by the unique in crystallo spectroscopy lab ‘Cryobench’ and its close relation with the protein crystallography beamlines at the European Radiation Synchrotron Facility.

There are two sides to this project, fundamental research and biotechnological development. First, we propose to use a combination of optical spectroscopy, mass spectrometry and X-ray crystallography to map the specific effects of singlet oxygen onto proteins. This will provide a framework to understand how a protein can sense or generate SO. We will then take advantage of several recently-designed protein systems capable of sensing or generating SO, improve them by semi-random mutagenesis and test their usability in vivo. Altogether, the results will help us understand on one hand how singlet oxygen affects proteins and on the other hand how SO can be tamed and exploited for beneficial uses.