Structural and dynamical properties in protein ligand interactions

• Strukturelle und dynamische Eigenschaften von Protein Liganden Interaktionen

Sarter, Mona; Fitter, Jörg (Thesis advisor); Stadler, Andreas (Thesis advisor)

Aachen : RWTH Aachen University (2020, 2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020

Abstract

In this work structural and dynamical properties of protein ligand interactions were analysed. This was done predominately by the use of scattering techniques. In addition to scattering techniques complimentary techniques were used to supplement the information obtained. The main scattering technique used was incoherent neutron scattering, as it allows for a separation of the analysed protein and its surrounding hydration layer. Two protein ligand systems were analysed. The first was the protein streptavidin(STV) and its ligand biotin (B). In addition to this, a genetically engineered Försterresonance energy transfer (FRET) based glucose sensor was analysed with and withoutadded glucose. For the selected systems, the specific challenge was to separate the influences of the hydration layer and the protein. Similarly, it is also challenging to create models that incorporate all the separate observations to form a cohesive interpretation. In order to regard the STV protein independently from the surrounding hydration layer neutronscattering was chosen as the appropriate technique. Whereas, for the glucose sensor thestructural changes were deemed to be the most important information to obtain and therefore X-ray scattering was employed. For the STV+B interaction it was considered that molecular dynamics play a vitalrole for the biological function of proteins. Changes in the proteins conformational entropy and of the hydration layer influence the binding process for many protein ligand interactions. Here this was investigated for STV, as well as the change in the internal dynamics of STV upon biotin binding. Quasi elastic neutron scattering (QENS) was used to investigate the change of conformational entropy of the protein and its dynamics. QENS results show that the conformational entropy of STV is reduced upon biotinbinding. Thermal diffusion forced Rayleigh scattering (TDFRS) results also indicatean increased entropy of the hydration layer. This indicates that the hydration layerplays an important role in stabilising the binding of biotin to STV. Comparing the change in conformational entropy per residue to that of other biological processes showed that in the STV+B binding process it is comparable to that of the transition from anunfolded myoglobin to a molten globule structure. When compared toother protein ligand interactions per residue it was found to be an order of magnitude larger. This indicates that within STV a significant change of conformational entropy occurs uponbiotin binding, more than can be explained by the already established conformational changes. Therefore, the internal STV dynamics before and after biotin were compared. This showed that the flexibility of streptavidin is greatly reduced upon biotin bindingleading to the complex being more rigid. The second system was a genetically encoded FRET-based biosensor, which was developed as a tool to quantify metabolites in nourishing solutions of living cells. The sensors consist of a central metabolite binding protein and flanking fluorescent proteins(FP) affixed by different linker sequences. FRET measurements are sensitive either to distance or orientation changes of FP domains as response to glucose binding. Size exclusion chromatography - small-angle X-ray scattering (SEC-SAXS) measurements have been performed to investigate, if either large-scale structural changes of the FP positions or relative orientation changes occur as response to glucose binding. Based on the measured SAXS curves modelling of the FP domains was performed. It was determined that glucose binding results in large-scale structural changes of the positions of linked FP domains for one of the sensors. These structures fit with the behaviour of the sensor expected due to fluorescent measurements. This was achieved by first regarding the hydration layer and the protein sepparately, by taking advantage of the sensitivity of neutrons to the scattering cross section of different hydrogen isotopes. In addition calorimetric techniques and TDFRS were used to support the neutron data obtained for the STV systems. In the case of the glucose sensor SAXS and FRET results were considered together.