Protein Characterization by Modern Laboratory Small-Angle X-ray Scattering

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch

In the living protein world, structure is tightly linked to function. Understanding the structure and interactions are therefore paramount, when characterizing protein systems. Malfunctions in the protein machinery can lead to diseases, and proteins play a vital role in pathological infections of hosts by microorganisms like bacteria. Elucidating the structure is therefore a key step towards discerning the protein function and also in the potential development of treatments targeting protein pathways or products. Solution small-angle X-ray scattering (SAXS) is a non-destructive technique that allows the study of proteins and soft condensed matter at the nanoscale under near-native conditions. In this thesis, four scientific SAXS projects are presented. First, a more technical work is presented on the modern flux-optimized laboratory SAXS instrument at Aarhus University with an in-line sample changer, in which the optimization parameters and an array of characteristic application examples have been accomplished to highlight the capabilities of the instrument for protein solution scattering. Next, three protein characterization projects are presented with diverse approaches of SAXS data analysis. The near-native solution structures of the protein RipA and truncated constructs from M. tuberculosis were determined through rigid-body refinement (RBR) analyses. The solution state of the N-terminal coil-coil stalk was identified as crossed dimers and for full-length RipA as monomers with the catalytic RipAc domain in its zymogenic state connected by a short flexible linker flexed back alongside the stalk. Efficient in-house RBR programs were developed in the process. A protein-protein interaction and enzyme positioning during cell division was proposed based on the findings. Time-resolved SAXS was used in the next work to study the fibrillation of FapC functional amyloid and truncated constructs from Pseudomonas bacteria. A modified analytical cylindrical model with elliptical cross section was combined with an experimental data set that accounted for an aggregated pre-fibrillar state. The expression was fitted to the time-resolved data yielding the ensemble-averaged solution structure of the formed fibrils over time. Changes in fibrillation patterns were detected between FapC wild type and the truncated constructs and the dimensions of the fibrillar cross sections suggested formation of fibril bundles. In the last project, low-density lipoprotein nanoparticles were structurally characterized as a function of temperature with a novel SAXS model of a flattened core-shell superellipsoid of revolution which contains three internal layers of cholesteryl esters at low temperature. A melting of the core layers was observed and reflected in the structural parameters, which shows a transformation into a spherical shape. The model was additionally applied to recent samples from healthy and pathologically diagnosed patients with myocardial infarction to build clinical structural profiles for healthy and sick individuals.
Original languageEnglish
PublisherAarhus Universiet
Number of pages206
Publication statusPublished - Apr 2019

Note re. dissertation

PhD defence, Wednesday, 27 March 2019 at 13.15<br/>Place: Building 1514, room 213, Lecture Theatre I, Department of Chemistry, Langelandsgade 140, Aarhus University, 8000 Aarhus C.<br/>Title of dissertation: Protein Characterization by Modern Laboratory Small-Angle X-ray Scattering<br/>Termination date: 04.04.2019

    Research areas

  • SAXS, Small-angle X-ray scattering, Proteins, Scattering, Radiation, Lipids, low-density lipoprotein, FapC, RipA, Thesis, PhD Dissertation

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