Mελέτη της διαμόρφωσης και της δυναμικής H-NOX (heme-nitric oxide/oxygen binding) επικρατειών και των συμπλόκων τους με σηματοδοτικά μόρια μέσω φασματοσκοπίας NMR ; Conformational study of H-NOX (heme-nitric oxide/oxygen binding) domains and their complexes with signaling molecules through NMR spectroscopy

Nitric oxide (ΝΟ) plays a key role in the the convertion of GTP to cGMP. The main receptor of NO is the sGC enzyme. sGC is a heterodimer, composed of α1 and β1 subunit, of which the latter contains the heme binding H-NOX domain, responsible for NO recognition and binding. NO binds upon the Fe of H-NOX and triggers electronic and conformational changes, leading to the signal transmission from the H-NOX domain to the catalytic and the subsequent enzyme’s activation. Impaired function of the NO/sGC/cGMP signaling pathway has been linked several diseases, such as hypertensionand heart failure. Low bioavailability of NO inhibits the pathway while heme oxidation makes sGC unresponsive to NO. As a central player in this axis, H-NOX is the focus of intense research efforts aiming to develop therapeutic molecules that enhance the pathway’s activity. Two classes of compounds have been described that directly affect the function of sGC: stimulators which activate sGC in spite of the low NO concentration and activators which replace oxidized heme, restoring the proper function of the enzyme. H-NOX domains have been identified not only in mammals but also in bacteria.In the present thesis, we present the conformational study of three bacterial H-NOX proteins, from Nostoc sp. (Ns H-NOX), Caldanaerobacter subterraneus (Cs H-NOX) and Vibrio cholerae (Vc H-NOX) and the mode of interaction with diatomic gases and chemical compounds. By applying NMR spectroscopy, we report the conformational exchange effect during the heme replacement of Ns H-NOX domain by the activators BAY 58-2667 and BAY 60-2770, leading Ns H-NOX to adopt a more rigid conformation. Same approach is applied to determine how the stimulator BAY 41-2272 interacts withNs H-NOX domain. Combination of tow different spectroscopic techniques, we study the conformational and electronic changes of the Ns H-NOX domain after the binding of NO. Site – directed mutagenesis experiments identify the role and the participation of the two tunnels of Ns H-NOX domain, in the entry ...