Development and Application of Proton-Detected Solid-State NMR Spectroscopy for Elucidation of Membrane Protein Function

• Applicant: Professor Rasmus Linser, Ph.D.
• Subject Area: Structural Biology ; Biophysics
• Term: from 2015 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270263529

Solid-state NMR is of ever-growing significance for characterization of structure and dynamics in solid proteins, most eminently amyloids and membrane proteins. Traditional methods have purely relied on non-proton resonances. Recently, as a significant complement, proton detection with intrinsically eightfold higher sensitivity compared with 13C has gained enormous interest. The availability of 1H chemical shifts in solid-state NMR owes to recent technical and methodological developments, for which my contributions have played a major role.We have employed protons for various now popular technical purposes. However, despite the ubiquitous role of protons/hydrogens in all cellular processes, accessibility of 1H information has hardly been exploited with respect to exploration of protein functionality. This is particularly true for membrane proteins, an important class of proteins among the main targets of solid-state NMR. Out of the innumerable biological processes hinging on the interplay of proteins with protons or hydrogens in some way, our focus is on two representative classes of membrane proteins, voltage sensors and integral-membrane proteases.Voltage sensors (VS) are elements of different ion channels and enzymes of broad significance particularly for regulating neuronal membrane potentials. We will subject a small, representative VS, of which atomic structure, voltage sensing, and 1H-conducting mechanism are largely elusive, including mutants expected to represent conducting or non-conducting states, to NMR methods characterizing proton behavior. As such, we will elucidate mechanistic features of proton conductance and the potential-dependent conformational changes in voltage sensing.Integral-membrane proteases are enzymes involved in several signaling pathways through regulated intra-membrane proteolysis (RIP). This common mechanism plays a prominent role in signaling, transcriptional regulation, for example of lipid biosynthesis, and in cleavage of proteins like the Amyloid Precursor Protein (APP). Focusing on a small, representative protease, we will assess questions of general significance, like domain mobility, substrate entry, and water channeling into the active site, buried in the hydrophobic space of the membrane.In both cases, understanding of the mechanism with atomic detail hinges on detailed information about solvent interactions with the water-exposed side chains, protonation states, trajectories, and physicochemical properties characterizing proton/water transfers in addition to the general structural and dynamics parameters in a lipid environment.Relying on my long-standing expertise in development and application of 1H-detected solid-state NMR and membrane-proteins, this case study towards characterization of protons in their biological context will pave the way for an unprecedented level of insight into basic membrane protein functionality and provide methodological groundwork for future structural biology.

• DFG Programme: Independent Junior Research Groups