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In and through the membrane by solid-state NMR: investigations of the human voltage-dependent anion channel 1 and Nup98-derived FG-domains

dc.contributor.advisorAndreas, Loren Dr.
dc.contributor.authorNajbauer, Eszter Éva
dc.titleIn and through the membrane by solid-state NMR: investigations of the human voltage-dependent anion channel 1 and Nup98-derived FG-domainsde
dc.contributor.refereeGriesinger, Christian Prof. Dr.
dc.description.abstractengMagic-angle-spinning NMR spectroscopy is a powerful tool for the investigation of protein structure, dynamics and interactions at atomic resolution. We have applied this technique to investigate two widely different systems: the human voltage-dependent anion channel 1, a 31 kDa membrane protein, and Nup98-derived hydrogel particles that recapitulate the selectivity of the nuclear pore complex. The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane, responsible for the transport of all ions and metabolites through the membrane. The channel derives its name from the drop in the channel’s conductance upon applied voltage. Although the protein had been previously extensively studied in a detergent environment, much less was known of its structure, dynamics and interactions in a native-like lipid bilayer. Using fast magic angle spinning (55 kHz) NMR spectroscopy on a 2D lipid crystalline preparation of human VDAC1 previously shown to yield solid-state NMR specta of exceptional resolution, we have significantly increased the assignment of the protein from 28% to 71%, and showed that the protein’s topology in a lipid bilayer is essentially the same as in a detergent environment: a 19-stranded β-barrel with an N-terminal kinked α-helix position inside the pore. Using a mutant implementing the channel’s closed state, we found that dynamics appear to be a key element in the protein’s gating behavior. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel in comparison to the N-terminal helix, and identified three binding sites on the C-terminal barrel wall. On the other hand, through investigation of a quintuple VDAC mutant implementing the channel’s closed state, we found that channel closure leads not only to destabilization of the the C-terminal barrel, but also of the α2 helix. We also observed an alternative mechanism for closing of the channel through steric blockage by the Bcl2-antisense oligonucleotide, G3139. In relation to this project we developed a method to probe the environment of a membrane protein using z-z mixing under conditions of fast spinning and perdeuteration. We devised 3- and 4 dimensional pulse sequences that allowed us to determine site-specific exposure of the protein to mobile water and lipids. We also determined transfer rates from protein to lipid and within the protein to explain site-specificity. The peak broadening caused by extensive drift during long multidimensional experiments made it necessary to find a way to compensate the effect of field drift. We found that a simple linear correction is sufficient to significantly improve spectral resolution. We achieved this using a script written in the C programming language, but directly executable from Topspin to correct for the drift occurring during 2D, 3D, and 4D experiments. Lastly, we investigated hydrogels formed from Nup98-derived FG-domains by a combination of biophysical and structural methods. Cohesive FG (phenylalnine-glycine)-repeat-domains phase separate into a condensed phase that forms the selective permeability barrier of nuclear-pore-complexes (NPCs). Sequence complexity stemming from the lengths (~600 residues) and sequence heterogeneity of native FG domains has so far limited nanoscopic insight. Here we overcame the challenge by utilizing a perfectly repetitive GLFG52x12 peptide as a model FG domain, dramatically reducing chemical complexity and thus enabling measurements down to amino-acid-level resolution using a combination of solution and magic-angle spinning NMR spectroscopy methods. Several further insights were gained through microscopic observations: 1) Although phase-separated FG repeats appear hydrogel-like, the protein remains nanoscopically mobile (ns timescale mobility of the peptide chain) and disordered, lacking secondary structure in both hydrogel and solution states. 2) Increasing salt concentration not only enhanced phase separation, but also slowed down the residue-specific backbone dynamics in the hydrogel phase. 3) The change of chemical shifts/ backbone dynamics upon phase separation suggest contacts involving phenylalanine residues, which were previously shown to be essential for phase separation. However, profound changes of hydrophobic leucine were also observed, and eliminating all leucine residues in the presence of phenylalanine strongly disfavored phase separation. Consistently, we observed a lower critical solution temperature (LCST) behavior of the protein, where increasing temperatures enhanced phase separation, suggesting that phase separation is driven not by enthalpic aromatic contacts, but by entropy. We constructed a phase diagram for the GLFG52x12 peptide under varying salt conditions from which we could quantitatively determine the enthalpy and entropy of phase
dc.contributor.coRefereeJanshoff, Andreas Prof. Dr.
dc.contributor.thirdRefereeGörlich, Dirk Prof. Dr.
dc.contributor.thirdRefereeDe Groot, Bert Prof. Dr.
dc.contributor.thirdRefereeEddy, Matthew Prof. Dr.
dc.subject.engSolid-state NMR spectroscopyde
dc.subject.engMembrane proteinde
dc.subject.engVoltage-dependent anion channelde
dc.subject.engMagic-angle spinningde
dc.subject.engPhase separationde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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