PROJECT SUMMARY Over half of drugs currently in use target integral membrane proteins (IMPs) because of the critical roles they play in linking the cell to its environment. For example, blood vessel epicardial substance (BVES), which has been linked to cardiac health, is among more than 300 IMPs implicated in human diseases, but with no structural data to assist in therapy or drug development. The increasing number of IMPs that have been implicated as putative drug targets in prevalent diseases drove the need for IMP structural studies to understand protein function and their role in disease, and to facilitate drug development. Nonetheless, the experimental determination of IMP structures remains challenging, as they make up only about 3.5% of protein structures currently housed in the Protein Data Bank (PDB). We propose a hybrid experimental and computational approach for IMP structure prediction. Herein, we develop an IMP structure prediction protocol that uses ROSETTANMR, a protein structure prediction program optimized for IMPs with limited NMR restraints. The approach uses paramagnetic tagging of non-natural amino acids (nNAAs) to collect NMR paramagnetic restraints. To accomplish this, we tackle the challenge of site-directed lanthanide ion labeling (SDLL) in a protein, and determine optimal labelling strategies. Goal 1 of the present proposal is to develop protocols for paramagnetic tagging and simultaneous collection of distance and orientational restraints for IMPs. NMR structural restraints obtained from lanthanide-based tags will be used by ROSETTANMR for IMP de novo structure prediction. We expect that paramagnetic restraints will guide the prediction to low-energy and experimentally valid conformations and significantly improve IMP structure determination. Thus, goal 2 is to improve IMP structure prediction in using only paramagnetic restraints. Ultimately, these optimized protocols can be applied to a range of larger IMPs. The experimental techniques developed here will be transferable to other IMPs (of biological importance) accelerating their experimental structure determination.