NMR at ultra-fast MAS (above 100 kHz) for the study of beta barrel and alpha helical membrane proteins.

• Applicant: Dr. Loren Andreas
• Subject Area: Structural Biology ; Analytical Chemistry ; Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397022504

The main objective of the research group is to further develop proton detected MAS at the highest magnetic fields and spinning frequencies currently available. The improvements in sensitivity and resolution available under these conditions will be applied to several membrane proteins, examples of which are detailed in the proposal, in order to determine the structure and dynamics for assemblies with characteristic size under about 50 kDa, with expected asymmetric size of about 10 to 30 kDa. We will utilize protons as sensitive reporters of structure along with isotopic labeling strategies in order to probe sites of particular interest in each system, whether the interesting biology involves ion conduction, small molecule interaction, or protein-protein interaction. Three systems have been selected for study: the beta barrel Opa60, the alpha helical ion channel E, and the alpha helical membrane protein M2. Selection of several proteins at the outset ensures a reasonable chance for successful structure determination should any one of the subprojects encounter difficulties. We propose to use NMR to measure the molecular structure of alpha helical and beta barrel membrane proteins at or near physiological conditions. Magic-angle spinning (MAS) is a powerful method to improve the resolution in NMR for preparations that do not undergo isotropic tumbling, such as lipid bilayer preparations of membrane proteins. Protons are the highest detection nucleus possible for proteins, a fact typically exploited in solution NMR. However, due to technical limitations, until recently, proton detected MAS was not broadly effective. This limitation is now removed with the introduction of a new generation of probes at high magnetic field. It is only recently that the combination of fast MAS, high magnetic field, and optimized sample preparation is realized to accelerate spectral acquisition and analysis for structural measurements. However, a fresh look at the techniques for recoupling and decoupling is needed, since many of the standard pulse programs are not operational at the new regime of MAS and magnetic field, while new possibilities are made possible by e.g high RF powers that are possible with small coils. The proposal is therefore composed of both method development and application to specific membrane proteins of biological interest, which will be used to demonstrate the methods.

• DFG Programme: Independent Junior Research Groups