The formation of biomolecular condensates by Liquid Liquid Phase Separation (LLPS) plays a key role in a manifold of cellular processes such as cellular organization, signal transduction and membraneless organelle formation. The biomolecular condensates formed are often complex in composition, but the underlying mechanism is rooted in thermodynamics. The demixing of a homogeneous solution into two phases, a protein enriched and a depleted phase, creates distinct environments for the proteins. These protein environments differ drastically in composition and viscoelastic properties. Thus, to decipher the cellular workings of biomolecular condensates, a biophysical toolbox to investigate these properties needs to be applied.

This thesis aims to develop methods to extract rotational dynamics and construct the full phase diagram of γD-crystallin in absence and presence of the cosolutes TMAO and PEG by utilizing electron paramagnetic resonance (EPR) spectroscopy. The results of the development of methods to determine rotational dynamics, a characterization of a novel site-specific label and the construction of the full phase diagram of γD-crystallin are summarized in three main publications.

The determination of rotational dynamics and the concomitant viscosity of phase-separated proteins is hindered by the inhomogeneous nature of the solution. In this first work we utilized continuous wave (cw) EPR in combination with site-directed spin labeling (SDSL) to follow the phase transition of γD-crystallin and extract the viscosity changes upon phase transition.1 We compared our findings with molecular dynamics (MD) simulations and fluorescence anisotropy measurements and could confirm our experimental EPR results.

In the second publication we characterized the novel spin label BASL with respect to its site specificity and reactivity on two model systems Bid and γD-crystallin.2 We could demonstrate that BASL displays an exquisite surface and site specificity, allowing an easy approach to orthogonal labeling and it is optimal for monitoring changes in rotational dynamics during the phase separation of γD-crystallin. We further showed that the distance distributions obtained by double electron electron resonance (DEER) spectroscopy are narrower for BASL compared to the conventionally available spin labels MTSL and MAP.

The last publication of this cumulative work is focused on the construction of the full phase diagram of γD-crystallin in absence and presence of cosolutes by utilizing a combined methodological approach including turbidimetry, centrifugation, pulsed EPR and Raman microspectroscopy. Thanks to the spatial resolution offered by Raman microspectroscopy we could construct the full phase diagrams and demonstrate that the addition of the cosolutes used increases the onset temperatures and reduces the critical concentration. We further highlighted the asymmetric shape of the phase diagrams. This work is a significant contribution in the endeavor to develop tools to determine the role of the environmental conditions on the phase separation of proteins.