B3.1: Time-resolved Solid-state NMR in Membrane Proteins in Lipid Cubic Phases

The PhD student of this research area will be qualified in connected areas of expertise: He/She will be trained in solid-state NMR and in particular in fast detection techniques, in membrane protein biochemistry, in optical methods required to control light-triggered reactions and in preparing and handling of photo-labile compounds. The combination of these areas of expertise brings them into the unique position to develop novel approaches for observing kinetic events within biological membranes.

The PhD project involves developing an approach in which biochemical events mediated by membrane proteins can be triggered by light and followed in a time-resolved fashion by solid-state NMR. Solid-state NMR and in particular MAS-NMR is the method of choice for membrane protein studies directly within the lipid bilayer. However, time-resolved studies are challenging due to limited sensitivity and novel approaches for light triggering have to be developed. The problem of sensitivity can be solved by fast detection schemes improving signal-to-noise per time and by the use of dynamic nuclear polarization. For light triggering, problems with optical density and mixing in liposome preparations have to be overcome. A solution to these problems is given by using lipid cubic phases (LCPs). In these phases, lipid bilayers, usually formed by monoolein, follow an infinite periodic minimal surface, which divides the space into networks of water channels. In contrast to liposomes, LCPs are optically transparent and all binding sites of embedded membrane proteins remain fully accessible. Therefore, the mixing step could be optimized by using caged compounds, which are activated within the LCP inside the MAS rotor.
This approach will be established for the membrane protein diacylgylcerol kinase (DAGK). DAGK phosphorylates diacylglycerol within the membrane through the transfer of the gamma-phosphate from ATP. The full enzymatic reaction can be detected conveniently by 31P-MAS NMR. The reaction will be triggered by the release of caged ATP, caged Mg2+ or by activating caged diacylglycerol.

MAS-NMR is usually performed on pellets of proteolipsomes (a). Membrane proteins can be also incorporated into lipid cubic phases (LCP), in which bilayers follow an infinite periodic minimal surface dividing the space into a network of water channels (b). Comparison of LCP and liposome samples containing the membrane protein DAGK (c). DAGK is a membrane embedded enzyme, which phosphorylates diacylglycerol at the expense of ATP (d).

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