Deciphering Interaction and Water Dynamics in Intrinsically Disordered Proteins with High Resolution Spectroscopic Tools

Artira Chowdhury
Despite its utmost significance in biology, water dynamics has often been the 'elephant in the room' in experimental biophysics, owing to the challenges in measuring it. Water dynamics becomes more significant in case of intrinsically disordered proteins (IDPs) as IDPs fall out of the classical 'structure-function' paradigm and thus the typical structure-function relationship is modified to a dynamics-function relationship for IDP recognition. No study so far touched the highly important and
more » ... plex topic on solvation dynamics on protein-protein interactions, let alone IDP recognition. In my PhD thesis my main aim has been to interrogate the interplay between solvation dynamics and binding mechanisms of IDPs involved in the nucleocytoplasmic transport pathway using a combination of steady state and picosecond resolved ensemble fluorescence spectroscopy and single molecule FRET (smFRET). I interrogated surface water dynamics in IDPs and its attenuation upon partner binding using three IDPs from the nucleocytoplasmic transport pathway, Nup153FG, NSP1FG and IBB which share one common binding partner, the nuclear transport receptor (NTR), Importinβ, despite having different binding mechanisms and sites. Nup153FG and NSP1FG belong to the class of IDPs known as FGNups, that constitute the permeability barrier of the nuclear pore complex (NPC). FGNups bind Importinβ through a set of ultrafast transient multivalent interactions retaining its disorder change while IBB forms a helix upon binding Importinβ. Solvent fluctuations in the dynamic Nup153FG-Importinβ complex and NSP1FG-Importinβ complex were unperturbed relative to the unbound state while in the IBB-Importinβ complex substantial relative slowdown of water dynamics was seen. These results directly showed a correlation between interfacial water dynamics and the plasticity of IDP complexes. Based on my results I concluded that solvation retardation poses energy barriers to conformational fluctuations, thus IDPs engage their partners with tunable solvation retardation, which directly correlates to their dynamics in the complex, enabling them to achieve functional diversity. In the context of nucleocytoplasmic transport such differential behavior of these two IDPs can be linked to their functions as well; dynamic FGNup-Importinβ complex likely expedites fast nucleocytoplasmic transport; while stable IBB-Importinβ warrants integrity of the import-complex during a transport event. Additionally I also performed femtosecond resolved fluorescence spectroscopy to explore the dynamics of IBB recognition by Importinβ across timescales spanning over 5 orders of magnitudes from 100fs to ~20ns. Capturing the entire timescale of the dynamics enabled me to look at the nature of these dynamics more closely. Contrary to several hypotheses reporting protein solvation dynamics to be governed by discrete timescales I found that the dynamics could be well explained by a power law type of relaxation suggesting a possible entanglement of the different timescales. This provides new insights into the nature of protein solvation dynamics in general as well in the context of IDP recognition. ii
doi:10.11588/heidok.00025353 fatcat:dgiwjolrx5ckrej3okdkgtjc64