In the last decades osteoporosis has become a major subject on the field of drug discovery and design. One of the enzymes recently considered important to use as a target for theses drugs is the enzyme H+-VO-ATPase. This proton pump is located in the osteoclast cells, which are positioned at the bone surface. These enzymes control the proton flux to the bone surface and consequently bone resorption. One major task on drug design is the knowledge of the secondary and tertiary structure of the enzyme under study. The topology of the V-ATPase protein complex has been largely established, however, only the three-dimensional structure of some individual subunits is known up to the time this thesis was printed. The work presented in this thesis focuses on the proton translocation channel located in subunit a of the V-ATPase complex. For this purpose, we designed two peptides, consisting of 25 and 37 amino acid residues, representing the seventh transmembrane segment of subunit a that encompass the proton translocation channel as well as the region of interest for possible inhibitors. Using a combination of NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy we analysed the conformation of these V-ATPase peptides in different membrane-mimicking environments: aqueous solutions of SDS (sodium dodecyl sulphate) and amphipols, and the organic solvent DMSO (dimethylsulphoxide). The conformation of the V-ATPase peptides in SDS micelles was studied by CD spectroscopy, however, due to their low solubility NMR spectroscopy turned out to be impossible. The CD results showed that the size of the peptide can drastically alter the solubilization in SDS. For the 37-residue V-ATPase peptide the overall conformation was a-helical and not dependent on the SDS concentration. On the other hand, the conformation of the 25-residue V-ATPase peptide depended on the peptide to SDS ratio changing between an a-helix and β-sheet conformation. As an alternative solubilising agent for the peptides in aqueous solutions we tested amphipols, a new class of macromolecules that were designed to solubilise transmembrane proteins for NMR and X-ray studies. The CD and tryptophan fluorescence spectroscopy results showed that both peptides aggregated in a β-sheet conformation. The formation of these b‑sheets aggregates might result from the interaction of the arginine residue present in the V-ATPase peptides with the anionic polymer. Such an interaction could prevent the peptide from crossing the hydrophobic core of the particle, preventing the formation of an a‑helix. High-quality high-resolution NMR spectra of the V-ATPase peptides were obtained in DMSO enabling to analyse the atomic structure of the peptides. The use of DMSO on structural studies of transmembrane polypeptides always raised some debate in the literature. This fact motivated us to perform a molecular dynamics study to investigate the solvation of the 25-residue V-ATPase peptide by DMSO. From this work, we show that DMSO can provide both polar and apolar environments to the peptide, making it a good membrane-mimicking organic solvent. The NMR study of the 37-residues peptide enabled us to confirm the a-helical conformation and to predict that the transmembrane spanning region of the seventh transmembrane segment is larger than expected. Instead of 25 transmembrane residues, we propose a transmembrane region of 32 residues. Furthermore, the NMR study of the 25-residue peptide lead us to postulate the existence of a hinge region located near the cytoplasmic end of the channel. It is proposed that the presence of this hinge allows the opening and closing of the proton translocation channel and provides flexibility for the channel to act as a binding pocket for inhibitors. The resulting NMR data sets for the two V-ATPase peptides are deposited in the BioMagResbank (BMRB) under the access numbers 15025 and 6878, and the calculated structure ensemble for the 25-residue peptide is deposited in the PDB databank with entry 2NVJ.
|Qualification||Doctor of Philosophy|
|Award date||8 Oct 2007|
|Place of Publication||[S.l.]|
|Publication status||Published - 2007|
- transmembrane proteins
- molecular conformation