Structure and dynamics of intrinsically disordered protein

Haris, Luman; Stadler, Andreas (Thesis advisor); Richtering, Walter (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

One focus of this dissertation is the investigation of structure and dynamics of intrinsically disordered proteins (IDPs) in the presence of the strong chaotrope guanidinium chloride (GndCl). For this purpose, small-angle X-ray scattering (SAXS) and neutron spin-echo spectroscopy (NSE) experiments have been performed. Two IDPs have been investigated in this approach: myelin basic protein (MBP) and prothymosin alpha (ProTa). Analysis of the SAXS data reveals a slight size reduction of MBP at low amount of GndCl, which is followed by re-swelling of the chain at cGndCl > 1 M. The shrinking is attributed to the anisotropic elongation that can be traced back to the secondary structure content within MBP and the change from a swollen and expanded chain to a more compact chain with Gaussian chain statistics. Meanwhile, NSE data were analyzed using the Zimm model with internal friction (ZIF) and the Zimm model with mode selection (ZMS): A significant contribution of internal friction was found in native and and slightly denatured MBP, which approaches a value of approximately 15 ns in MBP at high GndCl concentrations. The ZMS model reveals the presence of dynamically rigid elements in MBP that effectively cut of high frequency Zimm modes. This mechanism can be identified as being the source of internal friction in structurally expanded MBP. As comparison, we also studied the fully unfolded IDP ProTa. Here, we could confirrm structural collapse of ProTa at 1 M GndCl as seen previously with FRET. However, despite the structural collapse and due to the high negative net charge, the ProTa protein remains disordered regardless of GndCl concentration. The analysis of NSE spectra with ZIF model revealed the emergence of internal friction as a response to the presence of GndCl. Interestingly, similar to MBP, residual internal friction at high GndCl concentrations is also observed. The analysis of the NSE spectra with ZMS model has shown that internal friction influences the free energy landscape, thus effectively regulating binding and folding of proteins by locally restricting their range of motion. Finally, direct comparison to MBP has shown that the structural and dynamical change in IDPs occur within their respective disordered region. As the region expands, the internal friction reduces and saturates, allowing solvent friction to contribute more to the overall dynamics.The second aim of this work is to investigate the dynamics of the IDP COR15a and the folded globular protein myoglobin (Mb) in response to changes of hydration. For this purpose, quasielastic neutron scattering (QENS) experiments have been performed in D2O buffer and deuterated buffer containing 70% d8-glycerol. QENS experiments have been performed on neutron time-of-flight and backscattering spectrometers with different energyresolutions to probe a broad time range from the ps to several ns. The QENS experiments revealed slowing down of global diffusion and internal dynamics of COR15a and Mb in the presence of glycerol. The reduction of global protein diffusion of COR15a and Mb is similar as the change of translational diffusion of the buffer, which is an expected result and can be explained by the change of buffer viscosity. The reduction of internal dynamics is significantly larger for COR15a as it is for Mb when the buffer is changed from 0% d8-glycerol to 70% d8-glycerol. Concerning Mb, the reduction of internal dynamics is related entirely to the change of buffer viscosity, while for COR15a it appears to be caused by a combination of induced-folding of COR15a in 70% d8-glycerol and the higher solvent viscosity. Proper comparison with Myoglobin, especially in 70% glycerol, is difficult as the overall diffusion of Mb in 70% glycerol is very low and out of the resolution limit of the used instrument IN16B with the medium resolution BATS mode. The exact value of the global diffusion coefficient of Mb is important as input parameter for the fits of the QENS data of Mb. This open question will be addressed and resolved in the future using high resolution neutron backscattering spectroscopy. These experiments could not be performed during the PhD thesis as the research reactors have not been operational during the COVID19 pandemic.