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Journal article
Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions
Biophysical journal, v 123(23)
04 Nov 2024
PMID: 39497416
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Abstract
Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature TLLPS. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e., single-phase) and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich "droplets". Comparison of the experimental results with simulations of the sensitivity of 2D ssNMR crosspeaks to changes in populations of β-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%.Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature TLLPS. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e., single-phase) and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich "droplets". Comparison of the experimental results with simulations of the sensitivity of 2D ssNMR crosspeaks to changes in populations of β-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%.
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Details
- Title
- Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions
- Creators
- C Blake Wilson - National Institute of Diabetes and Digestive and Kidney DiseasesMyungwoon Lee - National Institutes of HealthWai-Ming YauRobert Tycko - National Institute of Diabetes and Digestive and Kidney Diseases
- Publication Details
- Biophysical journal, v 123(23)
- Publisher
- CELL PRESS; CAMBRIDGE
- Number of pages
- 18
- Grant note
- Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Wilson et al.
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Chemistry
- Web of Science ID
- WOS:001373650100001
- Scopus ID
- 2-s2.0-85209647580
- Other Identifier
- 991021955214504721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biophysics