Concentration Quantification of the Low-Complexity Domain of Fused in Sarcoma inside a Single Droplet and Effects of Solution Parameters

Abstract

Liquid–liquid phase separation (LLPS) is an important phenomenon in biology, and it is desirable to develop quantitative methods to analyze protein droplets generated by LLPS. This study quantified the change in protein concentration in a droplet in label-free and single-droplet conditions using Raman imaging and the Raman band of water as an intensity standard. Small changes in the protein concentration with variations in pH and salt concentration were observed, and it was shown that the concentration in the droplet decreases as the conditions become less favorable for droplet formation. The effect of exposure to 1,6-hexanediol was also examined, and this additive was found to decrease the protein concentration in the droplet. A model can be proposed in which the addition of 1,6-hexanediol reduces the protein concentration in the droplet, and the droplet disappears when the concentration falls below a certain threshold value.

Determining the Physico‐Chemical Composition of Biomolecular Condensates from Spatially‐Resolved NMR

“…The improved WET‐DPFGSE NMR experiment thus complements and extends previous NMR approaches that relied on the use of fluorinated molecules for quantification of protein or small molecule concentrations in phase‐separated systems [13, 17] . In addition, Raman microscopy can be useful to quantify protein concentrations, on the basis of the signals emerging from the different amounts of water present in the condensed and dilute phase [18] …”

“…[13,17] In addition, Raman microscopy can be useful to quantify protein concentrations, on the basis of the signals emerging from the different amounts of water present in the condensed and dilute phase. [18] The improved spatially-resolved NMR is applicable to effectively any IDP as long as its NMR resonances are observable. Protein concentrations determined by spatiallyresolved NMR characterize protein phase diagrams with high reliability.…”

Bestimmung der physikalisch‐chemischen Zusammensetzung von biomolekularen Kondensaten durch räumlich aufgelöste NMR

“…Das verbesserte WET‐DPFGSE‐NMR‐Experiment ergänzt und erweitert somit frühere NMR‐Ansätze, die sich auf die Verwendung fluorierter Moleküle zur Quantifizierung von Protein‐ oder kleinen Molekülkonzentrationen in phasengetrennten Systemen stützten [13, 17] . Darüber hinaus kann die Raman‐Mikroskopie nützlich sein, um die Proteinkonzentration zu quantifizieren, und zwar auf der Grundlage der Signale, die von den unterschiedlichen Wassermengen in der kondensierten und verdünnten Phase ausgehen [18] …”

“…[13,17] Darüber hinaus kann die Raman-Mikroskopie nützlich sein, um die Proteinkonzentration zu quantifizieren, und zwar auf der Grundlage der Signale, die von den unterschiedlichen Wassermengen in der kondensierten und verdünnten Phase ausgehen. [18] Die verbesserte ortsaufgelöste NMR ist auf praktisch jedes IDP anwendbar, solange dessen NMR-Resonanzen beobachtbar sind. Proteinkonzentrationen, die durch ortsaufgelöste NMR bestimmt werden, charakterisieren Protein-Phasendiagramme mit hoher Zuverlässigkeit.…”

Label-free autofluorescence lifetime reveals the structural dynamics of ataxin-3 inside droplets formed via liquid–liquid phase separation

“…The time evolution of droplets is difficult to be measured by NMR and EPR. Raman spectroscopy was employed to quantify the protein concentration in a single droplet in a label-free manner 34 , 44 , 45 and was recently used to observe the secondary structural changes in the C-terminal domain of TDP-43 associated with fibril formation 46 . However, the Raman signal is not always sensitive to slight changes in the molecular structure.…”