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Dynamics and Driving Forces of Macromolecular Complexes

dc.contributor.advisorGrubmüller, Helmut Prof. Dr.de
dc.contributor.authorBock, Larsde
dc.date.accessioned2013-06-05T07:42:03Zde
dc.date.available2013-06-05T07:42:03Zde
dc.date.issued2013-06-05de
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-001D-AFCE-4de
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-3864
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/
dc.subject.ddc570de
dc.titleDynamics and Driving Forces of Macromolecular Complexesde
dc.typedoctoralThesisde
dc.contributor.refereeGrubmüller, Helmut Prof. Dr.de
dc.date.examination2012-06-11de
dc.description.abstractengMany functions in living cells are governed by macromolecular complexes. To fully describe the underlying mechanisms, they have to be understood at atomic level. The present study combines data obtained by X-ray crystallography and cryo-electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Two functions of macromolecular complexes, the downregulation of neurotransmitter release by the SNARE protein complex under oxidative stress and the translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis, were investigated. First, the hypothesis that oxidation of two cysteines on linker of the SNARE protein SNAP-25B and consequent disulfide bond formation shortens this linker sufficiently to hinder complex formation was tested. For this purpose, MD simulations of the SNARE complex with and without the disulfide bond were compared. Disulfide bond formation lead to conformational changes of the linker and of three central hydrophobic layers necessary to form the SNARE complex. Previously, mutations of residues contributing to these layers have been shown to reduce neurotransmitter release, suggesting that the stability of these layers is crucial for complex formation. The results from the simulations agree with the hypothesis that disulfide bond formation leads to a destabilization of the SNARE complex thus rendering it dysfunctional. This mechanism is interpreted as a chemomechanical regulation to shut down neurotransmitter release under oxidative stress, which has been linked to neurodegenerative diseases. In a second part I investigated the ribosome, where after peptide bond formation, bound tRNAs translocate by more than 7 nm to adjacent binding sites (A and P to P and E), accompanied by large-scale conformational motions (L1-stalk, intersubunit rotation) of the ribosome. By combining existing cryo-EM reconstructions of translocation intermediates with high resolution crystal structures, we obtained 13 near-atomic resolution structures. Subsequently, MD simulations of were carried out for each intermediate state. The obtained dynamics within these states allowed to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. Rapid motions of the L1-stalk and the small (30S) subunit on sub-microsecond timescales were revealed, whereas tRNA motions were seen to be rate-limiting for most transitions. By calculating the interaction free energy between L1-stalk and tRNA, molecular forces were derived showing that the L1-stalk is actively pulling the tRNA from P to E binding site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving key protein-tRNA contacts. This explains how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements. Translocation is accompanied by rotations of the 30S ribosomal subunit of more than 20 degrees relative to the large (50S) subunit. For each translocation intermediate, the affinity of the two subunits with each other must be finely tuned enabling such conformational flexibility while maintaining integrity of the ribosomal complex. Analyzing the trajectories at residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and primarily located close to the axis of rotation. ii) contacts that are formed and ruptured depending on the rotation angle, seen mainly on the periphery. Strikingly, also these rotation specific contacts substantially contribute to the overall stability of the ribosomal assembly and are expected to maintain a constant interaction energy with low barriers for rotation. The simulations reveal that upon removal of tRNAs peripheral contacts are weakened and, in turn, intersubunit rotation angles decrease, in agreement with cryo-EM analysis of tRNA depleted ribosomes. The identified mechanisms for lowering free energy barriers and for fine-tuning affinities might have developed similarly in other macromolecular complexes.de
dc.contributor.coRefereeFicner, Ralf Prof. Dr.de
dc.subject.engmolecular dynamics simulationde
dc.subject.engSNAREde
dc.subject.engSNAP-25Bde
dc.subject.engoxidationde
dc.subject.engribosomede
dc.subject.engtranslocationde
dc.subject.engtRNAde
dc.subject.engintersubunit rotationde
dc.identifier.urnurn:nbn:de:gbv:7-11858/00-1735-0000-001D-AFCE-4-9de
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.identifier.ppn747818355de


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