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The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study
Biomolecular assembly processes based on liquid–liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-ju...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566631/ https://www.ncbi.nlm.nih.gov/pubmed/33057154 http://dx.doi.org/10.1038/s41598-020-74271-x |
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author | Cinar, Hasan Winter, Roland |
author_facet | Cinar, Hasan Winter, Roland |
author_sort | Cinar, Hasan |
collection | PubMed |
description | Biomolecular assembly processes based on liquid–liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-jump relaxation technique in concert with UV/Vis and FTIR spectroscopy as well as light microscopy to characterize the evolution of LLPS formation and dissolution in a time-dependent manner. As a model system undergoing LLPS we used the globular eye-lens protein γD-crystallin. As cosolutes and macromolecular crowding are known to affect the stability and dynamics of biomolecular condensates in cellulo, we extended our kinetic study by addressing also the impact of urea, the deep-sea osmolyte trimethylamine-N-oxide (TMAO) and a crowding agent on the transformation kinetics of the LLPS system. As a prerequisite for the kinetic studies, the phase diagram of γD-crystallin at the different solution conditions also had to be determined. The formation of the droplet phase was found to be a very rapid process and can be switched on and off on the 1–4 s timescale. Theoretical treatment using the Johnson–Mehl–Avrami–Kolmogorov model indicates that the LLPS proceeds via a diffusion-limited nucleation and growth mechanism at subcritical protein concentrations, a scenario which is also expected to prevail within biologically relevant crowded systems. Compared to the marked effect the cosolutes take on the stability of the LLPS region, their effect at biologically relevant concentrations on the phase transformation kinetics is very small, which might be a particular advantage in the cellular context, as a fast switching capability of the transition should not be compromised by the presence of cellular cosolutes. |
format | Online Article Text |
id | pubmed-7566631 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-75666312020-10-19 The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study Cinar, Hasan Winter, Roland Sci Rep Article Biomolecular assembly processes based on liquid–liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-jump relaxation technique in concert with UV/Vis and FTIR spectroscopy as well as light microscopy to characterize the evolution of LLPS formation and dissolution in a time-dependent manner. As a model system undergoing LLPS we used the globular eye-lens protein γD-crystallin. As cosolutes and macromolecular crowding are known to affect the stability and dynamics of biomolecular condensates in cellulo, we extended our kinetic study by addressing also the impact of urea, the deep-sea osmolyte trimethylamine-N-oxide (TMAO) and a crowding agent on the transformation kinetics of the LLPS system. As a prerequisite for the kinetic studies, the phase diagram of γD-crystallin at the different solution conditions also had to be determined. The formation of the droplet phase was found to be a very rapid process and can be switched on and off on the 1–4 s timescale. Theoretical treatment using the Johnson–Mehl–Avrami–Kolmogorov model indicates that the LLPS proceeds via a diffusion-limited nucleation and growth mechanism at subcritical protein concentrations, a scenario which is also expected to prevail within biologically relevant crowded systems. Compared to the marked effect the cosolutes take on the stability of the LLPS region, their effect at biologically relevant concentrations on the phase transformation kinetics is very small, which might be a particular advantage in the cellular context, as a fast switching capability of the transition should not be compromised by the presence of cellular cosolutes. Nature Publishing Group UK 2020-10-14 /pmc/articles/PMC7566631/ /pubmed/33057154 http://dx.doi.org/10.1038/s41598-020-74271-x Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Cinar, Hasan Winter, Roland The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title | The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title_full | The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title_fullStr | The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title_full_unstemmed | The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title_short | The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
title_sort | effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566631/ https://www.ncbi.nlm.nih.gov/pubmed/33057154 http://dx.doi.org/10.1038/s41598-020-74271-x |
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