This technology research and development project targets two major challenges for solid-state NMR and structural biology. First, sample handling represents a major bottleneck for solid-state NMR, costing time, money, and wasted sample. Powerful new solid-state NMR relax the volume and labeling requirements, but only if precious biological samples can be efficiently transferred into tiny MAS rotors. We will develop tools to make this process faster, more efficient and reliable. We will also develop tools to change sample conditions in the MAS rotor to better assess the functional state of a sample during experiments. Second, membrane proteins function in an asymmetric environment in the presence of transmembrane gradients of voltage, ions, and pH, but structural biology of membrane proteins is carried out in the absence of these gradients. This makes it difficult to correlate structure with function and creates one of the largest current knowledge gaps in structural biology. The project develops technology to perform NMR studies of membrane proteins embedded in lipid bilayers in the presence of transmembrane voltage, pH and ion gradients using several different approaches to ensure gradient stability during NMR data collection. We will also develop NMR-readout sensors to measure the membrane potential and pH and ion gradients during NMR data acquisition. This technology will enable direct measurement of the structure and dynamics of membrane proteins in the presence of gradients, revealing how transmembrane gradients regulate and drive membrane protein function. Since a large percentage of drug targets are membrane proteins that create, transform, dissipate or are regulated by transmembrane gradients, this technology will have high biomedical impact.