Finding the NMR based structure of Pmp3 Sample optimization and chemical shift assignment

Abstract

Pmp3 is thought to create a proton leakage through the plasma membrane. To get a better understanding of the function of Pmp3, it is important to determine the 3D structure of this protein. We used high resolution solution state NMR to determine the secondary structure of Pmp3. 15N,13C labeled Pmp3 was brought to expression in E.Coli as a his6-MBP-Pmp3 fusion protein. After Ni-NTA purification and gelfiltration the his6-MBP tag was cleaved off and Pmp3 was purified using TCA precipitation. Using DHPC as detergent high quality NMR spectra of Pmp3 were recorded. φ, ψ and the α-helices were predicted using the Chemical Shift Index and Talos+. Based on those predictions we propose a model for the structure of Pmp3 including two kinked trans-membrane helices.