| dc.contributor.advisor | Griesinger, Christian Prof. Dr. | |
| dc.contributor.author | Frischkorn, Sebastian | |
| dc.date.accessioned | 2019-09-27T09:07:18Z | |
| dc.date.available | 2019-09-27T09:07:18Z | |
| dc.date.issued | 2019-09-27 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0003-C1C4-5 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7654 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7654 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7654 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 571.4 | de |
| dc.title | A twist in NMR relaxation experiments: Application to the study of protein motions | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Griesinger, Christian Prof. Dr. | |
| dc.date.examination | 2019-06-04 | |
| dc.description.abstracteng | Understanding biological processes requires knowledge about the structure as well as dynamics of the involved molecules. Proteins are complex structures, which experience time
dependent changes due to atomic motions. The large number of atoms that compose proteins also cause a large variety of motions that can be observed in these macromolecules.
These motions range from femtoseconds to seconds and induce changes in the structure,
which might be pivotal for the functionality of a protein. Nuclear magnetic resonances
(NMR) spectroscopy provides a powerful tool to study protein motions, since it enables
the study of protein dynamics under physiological conditions and offers experiments that
cover large parts of the protein motion time scale. The first part of this thesis focuses on the study of protein motions that occur in the
microsecond to millisecond range. This regime of motions is targeted by so-called relaxation dispersion (RD) experiments. Here, the extreme CPMG (E-CPMG) experiment is presented, which combines the time scales of the two conventional RD experiments (CPMG and R1ρ) and allows a more accurate determination of fast kinetic processes by CPMG type experiments. Application of E-CPMG to study the folding/unfolding process of gpW protein in solution is presented. Previous studies have identified 68 residue gpW as an ultra-fast downhill folding protein that forms a α + β topology, which in solution stays in equilibrium with an unfolded β-hairpin conformation. Here, we show that the α-helices in gpW are also involved in this conformational exchange with a similar time scale as the β-hairpin. Furthermore, it is shown that residues in the α-helices are involved also in another much faster folding process. The RD profiles of these residues can only be described by a three-site exchange model and for the first time two distinct exchange
processes are detected in a single RD experiment. Complementary E-CPMG experiments of methyl side-chains as additional probe for structural changes, showed similar exchange kinetics as they were previously observed for the backbone of gpW β-hairpin region. A temperature dependent study of the slow exchange process lead to similar results for the activation energy reported by the different probes. These results indicate that global changes in the structure are involved in the formation of the β-hairpin region and also support the hypothesis of a hydrophobic collapse that assists its formation. Temperature dependent data of the folding kinetics reveal that the β-hairpin folding process of gpW in
solution can be explained by a two-state model with an energy barrier much larger than expected for a downhill folding protein. Thus, it can be assumed that the energy landscape of gpW in solution is more complex than it is described for a downhill folding protein. The second part of this thesis is dedicated to the study of protein motions that occur in the sub-τc regime by shuttle relaxometry. Shuttle relaxometry provides an alternative to the commonly used model free analysis for the study of local protein motions. The model free analysis is limited to high magnetic field strengths to ensure a high resolution and sensitivity, which are required for protein NMR. Using a motor-based shuttle system
the resolution of high magnetic fields (16.44 T) is combined with relaxation information from the stray field (10 - 0.5 T) of the NMR magnet. The aim of this work is to study the field-dependence of longitudinal relaxation rates and the effect of local protein motions on the Lorentzian behavior of the spectral density function. Initial experiments on ubiquitin showed a deviation from the expected mono-exponential decay for a flexible loop region as well as the C-terminal residues at magnetic field strengths below 4 T, indicating a stronger
influence of local motions at these low fields. During the course of this work intrinsic problems with the shuttle setup occurred and required relaxation data could not be acquired.
The effect of this intrinsic problems on the shuttle relaxometry data as well as the error diagnostics of the shuttle setup are described in detail. Additionally, relaxation data at several static magnetic fields was acquired to put the field-dependent R1 data from shuttle relaxometry into perspective. The results indicate the necessity of low-field relaxation data for a more accurate estimation of local protein motions and an improvement of the shuttle hardware. | de |
| dc.contributor.coReferee | Bennati, Marina Prof. Dr. | |
| dc.subject.eng | NMR spectroscopy | de |
| dc.subject.eng | Protein dynamics | de |
| dc.subject.eng | Relaxation Dispersion | de |
| dc.subject.eng | Shuttle Relaxometry | de |
| dc.subject.eng | gpW protein | de |
| dc.subject.eng | Ubiquitin | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0003-C1C4-5-9 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1677875577 | |