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Instrumentation and Methods Development for NMR of Oriented Biomolecules

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This thesis describes the design and construction of a three-channel (1H/13C/15N) switched-angle spinning solid-state NMR probe for a 500 MHz (11.7 T) magnet. The probe is designed for studies of membrane-associated proteins in native-like environments. This probe, which is the next generation of the pneumatic SAS probes built in the Martin Lab, keeps the pneumatic angle switching mechanism from the previous generation, while adding the third channel to enable the triple resonance experiments necessary for protein structure work. The channels utilize transmission line segments that act as tunable reactances, with the matching network for each frequency contained within an outer ground plane. The channels are capacitively coupled to the coil to enable smooth switching without bending the leads repeatedly. In order to study proteins with this probe, we will investigate the angular dependence of decoupling sequences. This is necessary because dipolar couplings are partially averaged out depending on the angle at which the sample is spinning. The angular dependence of popular decoupling sequences will be determined in order to assess how they change with the angle of the sample, enabling us to separately optimize for different angles within a single experiment. With SAS-optimized decoupling sequences, structural studies can be performed on membrane-associated proteins at different angles to extract further distance constraints and orientation information.

Also described is a high-throughput method for re-equilibrating predicted protein structures and evaluating them to predict their properties in order to search for targets with desired characteristics or other interesting features. This approach allows for the more efficient selection of protein targets for structure determination and biochemical characterization. This method has been used to investigate cocoonases from Heliconius butterflies and aspartic proteases from carnivorous plants, as well as other that are described elsewhere.

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