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Structure and Dynamics of Metal Ion Transporters using Solid-State Nuclear Magnetic Resonance at High Field and Fast Magic Angle Spinning

Final Report Summary - MEM-MAS (Structure and Dynamics of Metal Ion Transporters using Solid-State Nuclear Magnetic Resonance at High Field and Fast Magic Angle Spinning)

Proteins provide the basis for a multitude of important processes that support life. They are found as soluble proteins, such as enzymes, membrane associated proteins that interact with specific locations in the cell, and transmembrane proteins that often serve as the gatekeepers of cells and cellular compartments. Membrane proteins comprise 20 to 30 percent of all proteins, are vital for mediating entry and exit of molecules across membranes, signaling, and cell adhesion.
Determination of protein structures is the first step toward an understanding of protein function, and can aid in the rational development of small molecules that modulate function. Due to the paucity of membrane protein structures reported to date, the project set out to develop Nuclear Magnetic Resonance (NMR) spectroscopy methodology and apply that methodology to the determination of membrane protein structure.
We acquired spectra for two membrane proteins, one of which yielded well-resolved spectra amenable to structure determination, while the other membrane protein requires further sample optimization for high-resolution structural measurements. We have also developed new pulse sequences that accelerate the often time consuming step of resonance assignment. A method for robust side-chain resonance assignment was developed, which was used in a structure calculation of a protein dimer within a large viral capsid. We determined the structure of this viral nucleocapsid protein of previously unknown structure by application of new instrumentation that allows magic angle sample spinning (MAS) at 100 kHz. This represents a major improvement in the methodology, which should allow many more proteins to be efficiently investigated by NMR in the future. For us, the successful structure determination also served as proof that the method is successful for larger proteins with less structural homogeneity, and is currently being applied to a membrane protein of unknown structure in the lab in Lyon. Of particular interest to advancing the field, we demonstrated improvements to the sensitivity and reliability of the method by extending the measurements to 100 kHz MAS, which allows more nuclear spins to be observed while maintaining spectral resolution, and eliminating major bottlenecks for NMR based structure determination. These developments were critical to the de novo structure determination of the viral nucleocapsid protein, and are set to revolutionize the process of structure determination by solid state NMR.
Successful development of techniques that accelerate protein structure determination by MAS NMR is expected to allow a higher throughput, which will improve the understanding of membrane proteins, with extensive downstream societal benefits, such as the development of new treatments of human diseases.