Quantitative analysis of protein liquid-liquid phase separation

Numerous biological proteins tend to demix into a protein-rich liquid condensate phase and a protein-poor dilution phase. This process of liquid–liquid phase separation (LLPS) has emerged as a crucial mechanism for describing the production of biological condensates in live cells, which have been found to be critical to a number of biological functions and activities. In this thesis, I have focused on the quantitative studies of protein LLPS. First, a microfluidic platform was developed for high-throughput characterisation of protein condensate phase diagrams. It was implemented by combining the technology of droplet microfluidics, fluorescent imaging, and quantitative computational analysis. This new approach, replacing manual step-wise experiments, enables to generate phase diagrams with minimal sample and time consumption as well as high resolution. It may also facilitate the research of protein condensates such as the effect of small molecules and further quantitative physical studies. Next, a convenient protein condensate model system was explored, with tunable sizes and dispersity using microfluidics. It was achieved by transferring the condensate formation process into a droplet-microfluidic-based solid microgel formation step, separating liquefaction to condensate formation in a crowding agent. Furthermore, the protein LLPS mechanism was investigated from the perspective of colloidal interactions. I developed an inverse Monte-Carlo based method to interpret smallangle X-ray scattering data for the purpose of fitting interaction potentials using Derjaguin- Landau-Verwey-Overbeek (DLVO) theory. It was achieved by constructing a Monte-Carlo model that can produce simulated scattering curves from interaction potential parameters, with a surrogate machine learning model that may produce comparable results within shorter time periods, and finding the best fit in the given parameter space. For outlook, it may be enhanced with an extended DLVO theory to include hydrophobic interactions as a key step towards elucidating protein colloidal interactions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Qi, Runzhang

Advisor dc:contributor.advisor

• Knowles, Tuomas

• Liquid-liquid phase separation
• Microfluidics

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0002-7805-6501

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/347884