Structure determination of KCNE3 using paramagnetic restraints

The structures of membrane proteins are very difficult to determine by solution NMR since the slow tumbling of the protein-micelle complex results in severe line-broadening of the peaks in the spectrum. In addition, conventional NMR restraints for structure determination, such as NOEs, are extremely difficult to measure, especially for alpha-helical membrane proteins. To alleviate this problem we aim to introduce the measurement of paramagnetic restraints on membrane proteins. Paramagnetic restraints have been established for structure determination of soluble proteins but their use for membrane proteins is lacking behind. The measurement of these restraints such as Paramagnetic Relaxation Enhancements (PREs), Residual Dipolar Couplings (RDCs), and Pseudo-Contact-Shifts (PCSs) requires the introduction of a paramagnetic center into the protein. This will be achieved by attaching an EDTA-derived chelating agent onto a Cysteine residue, which will coordinate a paramagnetic metal ion, preferably of the lanthanide group. The protein chosen for this study is the 12 kDa protein KCNE3 that modulates potassium channel function.