Fluorescent proteins as nitric oxide sensors

The other idea of this work was to study globular proteins not only as nanostructures able to delivery reactive species, but also as biosystems able to monitor active molecules at cellular level.

In particular, the attention was focused on the development of fluorescent proteins able to monitor, through changes in their emission properties (intensity and/or lifetime), nitric oxide (NO^•) concentration.

Following this approach, an unexpected sensitivity for different fluorescent proteins has been studied and found.

A chimeric protein, CiNP-mTagBFP2, created by the fusion of Cimex Nitrophorin protein and blue fluorescent protein, was the first system to de investigated. In this case, the presence of NO^• causes a variation in the absorption spectrum that facilitates a Förster Resonance Energy Transfer (FRET)between the heme and the fluorescent proteins. It was possible to monitor the presence of this mechanism observing the reduction in the fluorescence intensity and lifetime of the fluorescent protein. Furthermore, the most interesting result was that this process was completely reversible. In fact, subjecting the protein to a nitrogen flow for about three hours, the fluorescence intensity and the fluorescence lifetime were brought back to the initial condition.

Observed the effect on the chimeric construct, the attention was focused on the single fluorescent proteins, mTagBFP2 and, subsequently, on other fluorescent mutated proteins, like TagRFP, EYFP, EGFP, Cys Free mTagBFP2, mTagBFP2 C26A and mCherryFP. The results underline that among all the proteins that have been studied, mTagBFP2 and TagRFP-T have proved to be the most sensitive to the presence of NO^•, both in neutral and in acid environment. Furthermore, this investigation highlights that NO^• dependence in lifetime and fluorescence emission can be related to S-nitrosylation of a Cysteine residue close to the chromophore. In fact, measurements on no-cystein proteins, mCherry and Cys Free mTagBFP2, confirm the lack of NO^• sensitivity.

Since mTagBFP2 has shown high NO^• sensitivity, preliminary analysis with live-cell fluorescence imaging were realised.

Using a FLIM set up, fluorescence lifetime images on HeLa cells transfected with mTagBFP2 and with mTagBFP2 C26A C114S C222S confirm the average lifetime measured in cuvette.

Furthermore, through fluorescence microscopy, the variation of fluorescence intensity in HeLa cells, transfected with mTagBFP2 and with the chimeric sensor, were monitored in time. With this set up, HeLa cells were subject to different NO^• concentrations that, as expected, reduced the fluorescence signals.

The NO^• induced, both in cuvette and in cells, a significant decrease in fluorescence lifetime and intensity suggesting a possible use of mTagBFP2, TagRFP-T and CiNP-mTagBFP2 as a biosensors for NO^•. However, contrary to the chimeric protein, for the fluorescent proteins, the nitrogen flow was not enough to bring the fluorescence lifetime and intensity back to the initial condition.

This result underlines that the mTagBFP2 as well as TagRFP-T are not sensors in the strict sense of the word, because the process is not reversible. More specifically: the fluorescent proteins are indicators of NO^•, not biosensors.

For the mTagBFP2 and TagRFP-T the process was reversible only with the addition of a reducing agent DTO, in micromolar concentration. Only in this case, the reversibility occurred and the fluorescence lifetime assumed values comparable to those obtained in absence of NO^•.

This last research shows a versatile potentiality of globular proteins usable as biosensors for the intracellular detection of NO^•.

Acknowledgments

Firstly, I would like to express my gratitude to Prof. Stefania Abbruzzetti and Prof. Cristiano Viappiani, who supervised me during these years. Their experience, patience, knowledge and continuous support were instrumental in increasing my skills and my scientific interest.

Furthermore, they gave me the opportunity to know different national or international research environments.

In this regard, I would like to thank Prof. Lorenzo Brancaleon who accepted me in his laboratory. During this long period at the University of Texas at San Antonio, I found a great group that with motivation, enthusiasm, and knowledge helped me during my stay in USA.

During my Ph.D I had also the opportunity to spend other two periods abroad, one at the Forschungszentrum Jülich and the other at the Medical University of Graz. In these occasions, Dr. Thomas Gensch and Prof. Roland Malli, respectively, welcomed me in their laboratories with great experience and hospitality. For this reason, I wish to thank them and their staff.

The STED and confocal experiments were performed at the Italian Institute of Technology of Genoa in close collaboration with the group of Prof. Alberto Diaspro, who hosted us several times in his laboratory. Particular thanks to Dr. Paolo Bianchini and Marco Cozzolino.

The femtosecond transient absorption spectroscopy measurements were performed at IFN-CNR Polytechnic University of Milan by the group of Prof. Giulio Cerullo, while the

photoinactivation test on bacteria cells and phosphorescence kinetics were performed by the group of Prof. Santi Nonell at Institut Quimic de Sarria Universitat Ramon Llull.

At the University of Parma, we collaborated with the group of Prof. Massimiliano Tognolini for the experiments involving eukaryotic cells and with the group of Prof. Silvia Bonardi for the experiments on bacterial photoinactivation.

Particular thanks are due to Prof. Stefano Bruno of the University of Parma for his knowledge and help in protein expressions and purifications.

I want to thank all the people of the bio-physics group at the University of Parma for the support, in particular my past and present lab colleagues: Pietro, Eleonora and Ludovica, always willing to help me.

Finally, I want to sincerely thank my family, Andrea and his family for their loving and support.

Fondazione Cariparma is kindly acknowledged for the financial support provided during my Ph.D and the University of Parma is kindly appreciated for the Overworld travel grant for my period at the University of Texas at San Antonio.