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

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.

Figure 1

Eight-channel in situ DLS setup: Side view (A) and top view (B). Laser and receiver optical elements are shown in a default position adjusted to the sample holder below located on a motorized stage.

Figure 2

Eight-channel in situ DLS setup (A) Portable hardware cabinet, including the diode laser, eight photomultiplier tubes (PMTs) for detection, autocorrelation units (ACUs) and a CPU (B) Setup of laser fiber and receiver optical elements aligned with the sample holder. A thermostat and heating foil below the holder is used for temperature regulation. The laser beam is schematically shown as dashed red line. (Approx. maximum height: 28 cm) (C) Schematic representation of the scattering geometry, measurement positions 1–8 (1 and 8 are labelled) and the eight-channel detection principle (D) Exemplary eight-channel DLS experiment using gold nanoparticles for setup verification. (E) Averaged radii (grey columns) as processed by the individual detectors. The values of the count rates, shown in parallel, approx. fit the expected decrease of scattered light with increasing distance from the focal point of the laser.

Figure 3

LLPS of BPTI (20 mg ml⁻¹), specifically triggered by SCN⁻. Variation of the temperature, the SCN⁻ concentration and the counter ion in individual drops (A–G) allows adjusting the number and size of LDCs in a defined area of the phase diagram. Scale bar: 20 μm.

Figure 4

Phase states and LLPS of GI (10 mg ml⁻¹) in the presence of 11% (w/v) PEG of different molecular weights (A–G). Images were recorded 20 min after mixing. Crystals formed in the presence of PEG 8′000 and PEG 10′000 (E–F). Scale bar: 40 μm.

Figure 5

Real-time monitoring of LDC formation applying multi-channel DLS: BPTI (A–B) and GI (D–E): Radius distribution plot of BPTI (A), comparing exemplary three data collection channels and GI (D), with protein only at 20 °C as well as after mixing BPTI with KSCN at 10 °C (B) and mixing GI with PEG 20′000 (E) respectively. The individual radius plots are indicating differences in abundancy of particle species depending on the position of the measurement at different time points of cluster formation (B/E). Micrographs showing LDCs of BPTI at 10 °C (C) and GI at 20 °C, 20 min after mixing (F).

Figure 6

Monitoring of the tertiary structure integrity during the clustering process using near-UV CD spectroscopy. The LLPS process of BPTI at 10 °C (A) and GI at 20 °C (C) is continuously monitored. Thermal denaturing of BPTI is shown for comparison (B). The tertiary structure of BPTI is changing upon a temperature decrease from 25 °C down to 5 °C, indicated by a slight ellipticity change in the tyrosine-sensitive region of the spectrum.