Mechanistic Inferences From Analysis of Measurements of Protein Phase Transitions in Live Cells

Ammon E. Posey, Kiersten M. Ruff, Jared M. Lalmansingh, Tejbir S. Kandola, Jeffrey J. Lange, Randal Halfmann, Rohit V. Pappu

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

Original languageEnglish
Article number166848
JournalJournal of Molecular Biology
Volume433
Issue number12
DOIs
StatePublished - Jun 11 2021

Keywords

  • DAmFRET
  • high-throughput
  • machine learning
  • nucleation
  • prion

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