title: Quantification of Molecular Interaction Strength in Protein Condensates by Brillouin Microscopy
Creators: name: Beck, Timon
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2025
Subjects: subject: Brillouin microscopy, phase separation, biomechanics, optics, quantitative phase imaging,

description: Biomolecular condensates have recently been identified as a crucial element of intracellular organization. These membraneless organelles fulfill various roles, for example, in cellular stress response or DNA damage repair. The basis for the formation of biomolecular condensates are molecular interactions, driving a solution of biomolecules from a mixed into a phase-separated state and back. These interactions also define the physical properties of such condensates, which are relevant for their physiological function and pathology. However, the experimental toolbox for the assessment of the physical properties of biological condensates and their underlying molecular interactions has not evolved as rapidly as the field of biological phase separation. The lack of robust quantitative methods poses a roadblock to the further development of the field. Only robust, dependable measurements provide a solid ground for a fundamental explanation of so far mostly qualitative observations. In order to address the shortage of experimental techniques, I used Brillouin microscopy to study the physical properties of different protein condensates in vitro. Brillouin microscopy is an emerging tool in the field of biomechanics that has been mostly used to investigate the elastic properties of cells and tissues so far. Here, I exploited the ability of Brillouin microscopy to investigate liquid materials. I established it as a novel tool to quantify average molecular interaction strength as well as the dissipative properties of protein condensates, represented by the Brillouin shift and the Brillouin linewidth, respectively. Furthermore, I used quantitative phase imaging to measure the refractive index of the condensates, giving access to their protein concentration and protein volume fraction. In contrast to many other experimental methods, these optical techniques do not require extensive assumptions or modeling of the system and work for liquid and solid materials. I investigated two different phase-separating proteins from the family of heterogeneous nuclear ribonucleoproteins, hnRNPA1 and the fused in sarcoma protein (FUS). I monitored the physical changes in the protein condensates in response to altered temperature and ion concentration, which have been shown to affect phase separation. Conditions favoring phase separation (e.g., low temperatures) increased Brillouin shift, Brilouin linewidth, and protein concentration. In contrast to solidification by chemical crosslinking, the physical aging process of FUS condensates had only a small impact on the Brillouin shift, which was consistent with the establishment of an elastic network within the condensates. This process was suppressed at a high ion concentration. Finally, I compared different variants of the hnRNPA1 protein featuring a broad range of different saturation concentrations. My experiments showed, that parameters with an impact on the phase separation behavior also alter the physical properties of the respective condensates. These results provide a new experimental perspective on the time-dependent physical properties of protein condensates and their sensitivity to temperature, solution conditions, and amino acid sequence of the respective protein.