A Study of Biomolecular Interactions in Kappa-Casein And Sleeping Beauty Transposase

This thesis investigates the model intrinsically disordered protein (IDP) κ-casein and the multidomain protein Sleeping Beauty (SB) transposase. The research is divided into three aims: (1) examining the self-association and translational diffusion of κ-casein, (2) studying the DNA binding affinity and nucleoprotein complex assembly of SB transposase, and (3) experimentally testing the computationally predicted structural model of the SB paired-end complex. Using advanced techniques such as pulsed-field gradient NMR and time-resolved FRET, we reveal that κ-casein exhibits continuous self-association, significantly impacting its translational diffusion. At low volume fractions, κ-casein self-associates, leading to macroscopic phase separation, while at higher concentrations, it forms labile gel-like networks. For SB transposase, we employ microscale thermophoresis to determine its DNA binding affinity to transposon direct repeats, providing crucial insights into transpososome assembly. Furthermore, we experimentally tested the predicted model of the transpososome complex in solution using FRET-based distance restraints. Our analysis identified discrepancies between the computational model of the paired-end complex and the experimentally derived inter-residue distances. These findings have broad implications for both basic science and applied biotechnology, offering potential advancements in gene therapy and genetic engineering, and highlighting the complex interplay between protein structure, function, and environment in elucidating IDP interactions. This research enhances our understanding of IDP behavior in crowded environments and contributes to optimizing transposon-based gene delivery systems.