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

doi:10.1016/j.heliyon.2019.e03016

A multi-channel in situ light scattering instrument utilized for monitoring protein aggregation and liquid dense cluster formation

Heliyon. 2019 Dec 13;5(12):e03016. doi: 10.1016/j.heliyon.2019.e03016. eCollection 2019 Dec.

Authors

Sven Falke^{1

2}, Hévila Brognaro^{1

3}, Arayik Martirosyan¹, Karsten Dierks⁴, Christian Betzel^{1

2}

Affiliations

¹ University Hamburg, Institute of Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Build. 22a, Notkestr. 85, 22607, Hamburg, Germany.
² The Hamburg Center for Ultrafast Imaging, c/o DESY, Luruper Chaussee 149, Hamburg, 22607, Germany.
³ Centre for Free-Electron-Laser Science, c/o DESY, Luruper Chaussee 149, Hamburg, 22607, Germany.
⁴ Xtal Concepts GmbH, Schnackenburgallee 13, 22525, Hamburg, Germany.

Abstract

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.

Keywords: Biochemistry; Biophysics; Bovine pancreatic trypsin inhibitor; Crystal nucleation; Glucose isomerase; Liquid dense clusters; Multi-channel dynamic light scattering; Nanotechnology; Patchy nanoparticles; Protein oligomerization.