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Characterization of structure, dynamics and inter- actions of the lipopolysaccharide- binding protein LBP, the hepatitis B virus X protein (HBx) and the unfolded-state ensemble of ubiquitin

Delgado, Leonildo. Characterization of structure, dynamics and inter- actions of the lipopolysaccharide- binding protein LBP, the hepatitis B virus X protein (HBx) and the unfolded-state ensemble of ubiquitin. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_13021

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Abstract

In this thesis, the structural ensembles and interactions of three proteins in different states have been characterized by high-resolution solution nuclear magnetic resonance (NMR) in combination with several other techniques: (i) the lipopolysaccharide (LPS)-binding protein LBP, which is the first receptor of LPS by the innate immune system, (ii) the hepatitis B virus X protein (HBx), which is involved in hepatitis B virus entry and replication, and (iii) urea-denatured ubiquitin, as a model system for an unfolded state ensemble.
Chapter 1 provides an introduction to the current state of Structural Biology, stating the importance of the three-dimensional structure of biomolecules and of changes in the structure during function. However, it is now recognized that also highly disordered proteins with no fixed three-dimensional structure, such as intrinsically disordered proteins (IDPs), have important functional roles. Related to this phenomenon is the question how a specific amino acid sequence determines the three-dimensional conformation of a protein.
Chapter 2 describes the successful production of the human lipopolysaccharide (LPS)-binding protein (hLBP) in insect cells and its biophysical characterization. By circular dichroism (CD) it was shown to contain a mixture of secondary structure elements that is similar to the solved mouse LBP (mLBP) structure. Using a growth medium supplemented by isotope-labeled algal extract (AE) and yeast extract (YE), the protein was 15N-labeled and further characterized by NMR. A 1H-15N-TROSY HSQC spectrum allowed to resolve 324 out of 473 expected resonances. In addition, 15N T and T relaxation experiments led to the determination of the rotational correlation time (τc) of hLBP, verifying its monomeric state in solu- tion. To characterize the interaction of hLBP with various glycolipids, two tryptophan residues on the N-terminal tip were used as a probe to assess the binding of glycolipid ligands. Upon binding, the internal quenching of the tryptophan fluorescence was released. This effect was more pronounced for LPS F515 and Lipid IVa than for smaller glycolipids. Additional surface plasmon resonance (SPR) experiments showed that hLBP binds to glycolipid aggregates with low micromolar affinity. Further, it was shown by SPR that LBP covers LPS micelles at a 1:3 LBP:LPS stoichiometry. However, only in the presence of the cluster of differentiation 14 (CD14) protein the LPS disaggregates.
Chapter 3 provides a detailed structural and dynamic characterization of the hepatitis B virus X protein (HBx). Using NMR, a nearly complete backbone and side-chain assignment was achieved. The analysis of secondary chemical shifts and 15N relaxation data showed that, despite being highly mobile on the nanosecond time scale, the protein contains four regions with slower backbone dynamics and propensity to form transient secondary structure elements (α-helices and β-strand). Remarkably these regions overlap with known functional motifs of HBx.
Chapter 4 describes a comparison of single-molecule Förster Resonance Energy Transfer (smFRET), NMR and small-angle X-ray scattering (SAXS) data to describe the urea-denatured state of ubiquitin. The analysis showed perfect agreement between the dis- tance-distributions derived from smFRET and NMR/SAXS-restrained ensembles. The NMR measurements supplied important details on local structural propensities and backbone dynamics, and provided even short- and long-range interactions that are only populated to a low percentage. The SAXS data complemented the NMR data with additional constraints of the overall shape of the unfolded-state. The smFRET provided subpopulation-specific distance distributions over a wide range of denaturant concentrations and revealed chain reconfiguration times in the 50-100 ns range. Overall, the combination of the three methods presents the currently most comprehensive description of the structural and dynamic properties of an urea-denatured protein.
Advisors:Grzesiek, Stephan and Maier, Timm
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
UniBasel Contributors:Grzesiek, Stephan and Maier, Timm
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13021
Thesis status:Complete
Number of Pages:1 Online-Ressource (xiv, 106 Seiten)
Language:English
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Last Modified:07 May 2019 04:30
Deposited On:06 May 2019 09:38

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