Small angle scattering as a tool to study protein structure and interactions

Abstract: This thesis uses small angle X-ray and neutron scattering (SAXS/SANS) to gain structural and functional insight into the molecular regulation of critical life processes in prokaryotic and eukaryotic species. The presented studies highlight the strength of combining low-resolution structure determination with biophysical and in silico modelling methods to extensively characterize proteins and their interactions.  DNA-binding: MexR protein belongs to the family of bacterial transcription regulators and control the expression of multidrug efflux pumps in Pseudomonas Aeruginosa by binding to a DNA region of the operator. SAXS/SANS data supported by MD (Molecular Dynamics) simulations demonstrated that the MexR dimer in solution undergoes a DNA-binding conformational selection mechanism. To gain a better understanding about the system, a low-resolution structural model was resolved in order to assess protein binding to the entire operator region comprising of two closely located DNA recognition sites. The study demonstrates that the use of scattering techniques to investigate similar systems is straightforward and provides knowledge of relevance for clinical understanding and future drug design.  Viral host factors: Picornaviruses represent a large family of small RNA viruses that are responsible for a range of diseases in humans and animals. Recently a non-essential human phospholipase PLAAT3 was identified as a key host factor for some picornaviruses. Several picornaviruses representing different branches of the picornaviral phylogenetic tree contain a type of 2A protein in their genome that share a conserved H-box/NC motif with PLAAT3. To understand the role of these 2A proteins in the viral life cycle and to map their plasticity, high resolution techniques were complemented with SAXS to evaluate the structural rearrangements and flexibility.  Ubiquitination: In eukaryotes, ubiquitination is a fundamental posttranslational modification, where a small protein ubiquitin is covalently attached to a target protein via sophisticated multienzyme process. SANS can be used to study this mechanism in solution by modular deuteration of ubiquitin complexes. To explore this possibility further, an E2 conjugating enzyme was attached to a deuterated ubiquitin via an isopeptide bond, and a neutron contrast variation experiment was performed. To investigate the flexibility of the E2~Ub conjugate, a multi-state modelling approach was employed to sample its conformational landscape.  SANS methods in protein science: A final methods paper outlines and details the experimental requirements, procedures and pre-studies that need to be considered to optimise a successful experimental approach for SANS with contrast variation on biomolecular complexes and assemblies in solution. 

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