A multi-channel in situ light scattering instrument utilized for monitoring protein aggregation and liquid dense cluster formation
Sven Falke, Hévila Brognaro, Arayik Martirosyan, Karsten Dierks, Christian Betzel
Liquid-liquid phase separation (LLPS) phenomena have been observed in vitro as well as in vivo and came in focus of interdisciplinary research activities particularly aiming at understanding the physico-chemical pathways of LLPS and its functionality in recent years. Dynamic light scattering (DLS) has been proven to be a most efficient method to analyze macromolecular clustering in solutions and suspensions with diverse applications in life sciences, material science and biotechnology. For spatially and time-resolved investigations of LLPS, i.e. formation of liquid dense protein clusters (LDCs) and aggregation, a novel eight-channel in situ DLS instrument was designed, constructed and applied. The real time formation of LDCs of glucose isomerase (GI) and bovine pancreatic trypsin inhibitor (BPTI) under different physico-chemical conditions was investigated in situ. Complex shifts in the particle size distributions indicated growth of LDCs up to the μm size regime. Additionally, near-UV circular dichroism spectroscopy was performed to monitor the folding state of the proteins in the process of LDC formation.
Circular dichroism, Tertiary structure, Thermal stability, Protein folding, Chemical stability, Aggregation, Biochemistry