Solution NMR-based characterization of the structure of the outer mitochondrial membrane protein Tom40 and a novel method for NMR resonance assignment of large intrinsically disordered proteins
von Xuejun Yao
Datum der mündl. Prüfung:2013-10-23
Betreuer:Prof. Dr. Markus Zweckstetter
Gutachter:Prof. Dr. Markus Zweckstetter
Gutachter:Prof. Dr. Kai Tittmann
EnglischThis dissertation is composed of two parts. In the first part, the characterization of the structure of liposome-embedded Tom40 using a novel hydrogen/deuterium (H/D) exchange method is described. In the second part, a new high-dimensional NMR experiment for the automatic backbone resonance assignment of intrinsically disordered proteins is described. The Tom40 protein is the central component of the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 forms the conductive channel for translocation of proteins into mitochondria and has been proposed to fold into a β-barrel. As part of my PhD thesis, I developed an H/D exchange protocol to characterize the structure of integral membrane proteins embedded into liposomes. The H/D exchange protocol consists of two steps of H/D exchange followed by NMR spectroscopic measurement of the denatured monomer. Application of the method to liposome-embedded Neurospora crassa Tom40 (ncTom40) showed that the N- and C-terminal tails are disordered. In addition, slow solvent exchange provided experimental support for several β-strands in the predicted barrel region and an α-helix N-terminal to the barrel. Evidence was also provided for the presence of conformational instability in the first three N-terminal β-strands, which might be involved in Tom40 oligomerization and interaction with other TOM subunits. In addition, NMR-based titration analysis of a fragment comprising the N-terminal disordered part of ncTom40 with presequence revealed multiple presequence binding sites on Tom40 that may facilitate Tom40 binding to unfolded precursor proteins transiently and subsequent transfer of the preproteins into the translocation pore. Our interaction study of Tom40 and DHPC micelle further identified several hydrophobic clusters on Tom40, which could be the initial lipid binding sites during Tom40 folding into the membrane. In the second part of my work, I developed a high-dimensional NMR experiment named 6D HACACONCAH APSY to facilitate the assignment of intrinsically disordered proteins (IDPs). The sequence-specific assignment of IDPs is challenging mainly due to severe chemical shift degeneracy of IDPs. Moreover, conventional resonance assignment based on amide proton detection is complicated due to fast exchange of amide proton signals in HN-detected NMR spectra. Automated Projection Spectroscopy (APSY) enables the measurement of very high-dimensional NMR spectra by simultaneous chemical shift evolution of several nuclei, whereas NMR experiments based on alpha proton (HA) detection is able to avoid the line broadening due to solvent exchange. Our new APSY experiment which combines APSY and HA-detection correlates chemical shifts from six dimensions, providing an excellent resolution to decrease NMR signal overlap. The chemical shift correlation between one Cα-Hα group and its preceding one obtained from 6D HCACONCAH APSY can be directly used as sequential connectivity for automatic assignment. Application of this novel method to two typical unfolded proteins, 140-residue α-synuclein and 352-residue Tau, resulted in the reliable assignment of more than 70% of assignable residues. The spectral measurement and assignment calculation were finished in 3 to 4 days, highly reducing the overall analysis time and tedious manual assignment procedures. Our method is ready to be used as a quick and reliable assignment protocol for IDPs even with large molecular weight.
Keywords: NMR spectroscopy; Membrane protein; Intrinsically Disordered Proteins; hydrogen/deuterium exchange; Tom40