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13C sparse labeling for solid-state NMR investigations of biomolecular systems

dc.contributor.advisorLange, Adam Dr.
dc.contributor.authorFaßhuber, Hannes Klaus
dc.title13C sparse labeling for solid-state NMR investigations of biomolecular systemsde
dc.contributor.refereeLange, Adam Dr.
dc.description.abstractengThis thesis refers to 13C sparsely labeled strategies, including protein structure characterization on two different biomolecular systems, to obtain structural information via 13C-13C and 13C-15N correlations by solid-state nuclear magnetic resonance (ssNMR). By applying [1-13C]- and [2-13C]-glc labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in ssNMR spectra can be achieved for two different precipitation conditions (MPD and PEG). This allowed spectral resonance assignment in a straightforward manner and the collection of an unprecedented wealth of long-range distance information. High precision solid-state NMR structures of microcrystalline ubiquitin with a backbone root mean squared deviation (rmsd) of 0.7 Å were calculated in both conditions. A backbone accuracy of 1.57 Å (MPD) and 1.88 Å (PEG) to the concerning X-ray structures could be calculated. In the comparison between the lowest energy structures of the two systems one can declare that the 3D fold of Ubiquitin is identical. A global backbone rmsd value of 1.63 Å is calculated (residue M1-70V). Small site specific conformational deviations can be identified in the regions (L8-T12, D21, E34-I36 and for E51-G53). Interestingly, one can resolve similar structural heterogeneity in both crystallization conditions. For the MPD system structural heterogeneity is present for β-strands β1, β2, β3 and β5 as well as for the loop regions β1-β2 and α1-β3. In the PEG condition one can distinguish structural heterogeneity for the first and second β-strand β1, β2, residue I23, at the tip of the α-helix α1, residue D39, the third β-strand β3, residue L50, residue I61, the fifth β-strand β5 and residue L69. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that were found to be involved in conformational dynamics of ubiquitin on the ns to µs time scale, as reported in recent residual dipolar coupling (RDC)-based measurements and relaxometry experiments. We suggest that the conformational sampling of ubiquitin manifests itself as structural heterogeneity during the crystallization process. The second isotope labeling strategy is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are only 13C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the Type-Three Secretion System needle of Shigella flexneri, where 33 unambiguous distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved. This method can be applied for studying protein properties within large biological
dc.contributor.coRefereeBennati, Marina Prof. Dr.
dc.contributor.thirdRefereeKuhn, Lars Thorsten Dr.
dc.subject.engsolid-state NMRde
dc.subject.eng13C sparse labelingde
dc.subject.engprotein structure determinationde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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