| dc.description.abstracteng | Membrane proteins are an essential part of any living organism. They are cellular switches that trigger metabolic cascades regulating the cell metabolism, including cell division, gene expression and others functions as needed. Despite the tremendous efforts put in place to unveil the structural details of membrane proteins, there are a few techniques available to inform on the structure-function relationship in a physiological context. Proton-detected solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides unique abilities to address these challenging systems in near-native environments. However, there are still fundamental limitations such as the strong proton-proton dipolar couplings limiting high proton resolution. Although per-deuteration increases resolution, it comes with loss of structural information since only exchangeable amide protons are observed in the NMR spectra. Full protonation demands the fastest magic-angle spinning probes and the highest magnetic fields.
There are still many opportunities to improve solid-state NMR methods. We demonstrate that proton detected ssNMR under fast MAS can be used to map the membrane-surroundings in highly perdeuterated proteins in two independent systems. The membrane insertion of the alkane transporter, AlkL, from Pseudomonas putida and the human voltage dependent anionic channel, hVDAC, was investigated by proton detected solid-state NMR under 55 kHz MAS. This study showed that proton spin diffusion is sufficiently quenched using a perdeuterated protein with 100% back-exchange of amide protons at 55 kHz MAS. Having the dipolar network quenched allows for site specific information using proton-proton z-mixing experiments. And, we additionally introduce alpha proton exchange by transamination (α-PET), a novel method which consists of re-introducing Hα backbone protons while maintaining other protein sites highly deuterated. By applying α-PET, to both a microcrystalline α-spectrin Src-homology 3 (SH3) and a lipid reconstituted hVDAC samples, we showed an improvement in the NMR proton line widths with respect to the fully protonated samples by almost a factor of two. This allows for facile Hα assignment as well as unambiguous Hα-Hα long-range distances adding restraints for structure calculation at 55 kHz MAS. In addition, α-PET allows protein expression in protonated media which overcomes exchange limitations for the amide sites often seen in membrane systems.
We applied proton detected ssNMR under fast and ultra-fast MAS to two distinct membrane proteins in membrane environments. To begin, we investigated Mic10, a double pass transmembrane helical protein that adopts a hairpin-like structure in the inner mitochondrial membrane (IMM) involved in the formation of the cristae junctions. Our data indicated that the second transmembrane domain of the protein undergoes a conformational transition, which we hypothesize to be, in part, involved in the modulation of the IMM. To terminate, we investigated matrix protein 2 (M2). M2 is a homo-tetrameric membrane proton channel from influenza A. Using a fully protonated M2 protein in combination with ultra-fast MAS (105 kHz), we for the first time obtain complete assignment of the important residue tryptophan 41 and histidine 37 (H37). The proton assignment allowed us to unambiguously assign the τ tautomer in DPhPC membranes at pH 7.8 and to identify the hydrogen bonded arrangement of the key residue H37 by detecting a 2JNN inter imidazole-imidazole J coupling. In addition, real-time NMR measurement at low temperature allows determination of a high energy barrier of ~130 kcal/mol for rimantadine (rmt) pore binding which is consistent with the structural rearrangement of M2 upon pore binding previously proposed. Furthermore, we found that rmt pore binding disturbs the imidazole-imidazole H-bond. Finally, the location of a structured pore water hydrogen bonded to Nδ1 of H37 was identified at high pH by combining low temperature NMR, DNP and DFT calculation.
Altogether, in this thesis, we first introduce two novel methods for studying membrane proteins by proton detected ssNMR under fast MAS (55 kHz). Secondly, on our more applied approaches, we unveil specific structural characteristics of both membrane proteins, Mic10 and M2, in the relevant context of lipid bilayers. These pieces of evidence further support the necessity of carrying out structural studies in close to near-native conditions since the environment might play an important role in the structure and the function as well. | de |