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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
 
Final Report Year 2024

Final Report Abstract

Development of pharmaceuticals requires Angstrom scale structural information about the protein targets. This is particularly challenging for small membrane proteins, since they are particularly sensitive to sample conditions. We proposed to develop and apply magic-angle spinning NMR spectroscopy to study the structure and structural dynamics of membrane proteins, and have fully succeeded in doing so: We determined the structure of three beta barrel proteins. The first, AlkL, is involved in transport of ‘greasy’ hydrophobic molecules and is of interest for the engineering biocatalytic reactions within defined compartments. Using NMR, we addressed a controversy regarding structuring of extracellular loops that are important in molecular transport. The second, VDAC, is responsible for metabolite transport in humans and other eukaryotes, and is linked to important cellular events such as apoptosis. By assembling samples where VDAC is embedded in the native context of lipid bilayers, we could confirm the structure found before in detergent solution or detergent-containing crystals. The third, Opa60 from pathogenic bacteria, is thought to function by allowing the bacterium to attach to the host. We proposed to find out if the structure of the protein in lipid bilayers could help explain the attachment function of the protein. We were successful in determining the structure, and found that the extracellular loops of the protein are highly flexible, implying a complex mechanism for attachment. We are also investigating viroporins, which are typically oligomeric alpha helical proteins that accelerate conduction of ions across membranes. We studied the structure of the viral protein, M2, that is a drug target and in focus due to the prevalence of resistant variants of Influenza. In this case we uncovered particular protein-protein interactions that are only seen when the protein is assembled in the native context of lipid bilayers. This interaction helps to explain the function of the protein, namely, that the virus can detect a low pH environment and trigger infection. These results lay a foundation from which the development of improved therapies are expected to follow. They also establish magic-angle spinning NMR as an approach to study the structure of viroporins. We are now initiating investigations of protein E from SARS-CoV-2, which assembles together with a host cell protein that we think is important in answering whether the protein is a dimer or a pentamer.

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