Biophysics Seminar- “Measuring bridging forces in protein-DNA condensates” with Vikhyaat Ahlawat (Zhou Lab) and “ATP Converts Protein-Nucleic Acid Aggregates into Liquid Condensates” with Divya Kota (Zhou Lab)
Biophysics Seminar
January 17, 2025
2:00 PM - 3:00 PM
Location
SES 2214
Calendar
Download iCal FileMeasuring bridging forces in protein-DNA condensates
Vikhyaat Ahlawat
Zhou Lab, Department of Chemistry
Protein-DNA condensates mediate transcription and regulate gene expression and DNA repair. The intermolecular bridging forces stabilizing condensates have direct roles in these processes. Here we use optical tweezers to measure bridging forces. In the presence of protamine, a single condensate is observed on a 20.5-knt single-stranded DNA (ssDNA) tethered between two microbeads. Stretching produces force curves with a sawtooth pattern, suggesting that the condensate is dissembled by the sequential rupture of individual protamine-ssDNA bridges. The
bridging forces are 11.3 ± 4.6 pN, with distances of 1.3 ± 0.8 µm between adjacent bridges along the ssDNA contour. In contrast, double-stranded DNA (dsDNA) forms protamine-mediated tangles that can withstand forces high enough (~55 pN) for strand separation. ssDNA tracks unpeeled at nicks on dsDNA by overstretching seed tangle formation upon retraction, but the initial condensates have a sufficient ssDNA-to-dsDNA ratio to appear liquid-like, as indicated by a sawtooth pattern in the subsequent stretching. The presence of dsDNA raises bridging forces to 34 ± 8 pN, which are reduced to ~10 pN by the addition of external ssDNA. In line with these single-molecule results, protamine-dsDNA mixtures form solid-like aggregates and require the addition of ssDNA to become liquid-like droplets. The fusion speed of protamine-ssDNA droplets is slowed by the addition of dsDNA. This work not only demonstrates the first measurements of bridging forces but also shows that their magnitude in protein-DNA condensates can be tuned by the ssDNA-to-dsDNA ratio.
Zhou Lab, Department of Chemistry
Protein-DNA condensates mediate transcription and regulate gene expression and DNA repair. The intermolecular bridging forces stabilizing condensates have direct roles in these processes. Here we use optical tweezers to measure bridging forces. In the presence of protamine, a single condensate is observed on a 20.5-knt single-stranded DNA (ssDNA) tethered between two microbeads. Stretching produces force curves with a sawtooth pattern, suggesting that the condensate is dissembled by the sequential rupture of individual protamine-ssDNA bridges. The
bridging forces are 11.3 ± 4.6 pN, with distances of 1.3 ± 0.8 µm between adjacent bridges along the ssDNA contour. In contrast, double-stranded DNA (dsDNA) forms protamine-mediated tangles that can withstand forces high enough (~55 pN) for strand separation. ssDNA tracks unpeeled at nicks on dsDNA by overstretching seed tangle formation upon retraction, but the initial condensates have a sufficient ssDNA-to-dsDNA ratio to appear liquid-like, as indicated by a sawtooth pattern in the subsequent stretching. The presence of dsDNA raises bridging forces to 34 ± 8 pN, which are reduced to ~10 pN by the addition of external ssDNA. In line with these single-molecule results, protamine-dsDNA mixtures form solid-like aggregates and require the addition of ssDNA to become liquid-like droplets. The fusion speed of protamine-ssDNA droplets is slowed by the addition of dsDNA. This work not only demonstrates the first measurements of bridging forces but also shows that their magnitude in protein-DNA condensates can be tuned by the ssDNA-to-dsDNA ratio.
ATP Converts Protein-Nucleic Acid Aggregates into Liquid Condensates
Divya Kota
Zhou Lab, Department of Chemistry
ATP is best known as the energy source of cells; for this and other classical roles only micromolar concentrations are required and yet cells maintain millimolars of ATP. One proposed reason for the high levels is that ATP solubilizes protein aggregates by acting as a hydrotrope. Here we investigate a different reason. Our previous study demonstrated that ATP mediates the phase separation of positively charged (or basic) intrinsically disordered proteins (bIDPs). The present study shows that ATP converts various bIDP-nucleic acid aggregates into liquid droplets. ATP displaces the nucleic-acid component and takes millimolar concentrations to outcompete the latter. Under static conditions, it may take many minutes for ATP to complete the displacement; this process is sped up enormously when ATP is supplied in a flow. The fusion of ATP-converted droplets can be slower than the bIDP-ATP droplets, and the slowdown is due to residual nucleic acids inside droplets. In-cell experiments confirm the in vitro effects. Protamine-DNA aggregates are taken up by cells; upon the addition of external ATP, DNA is displaced by ATP from the aggregates and the resulting condensates appear to be liquid. Overall, our findings indicate that the mM ATP levels in cells may be required for keeping IDP-nucleic acid condensates in a liquid state and explain why ATP depletion turns membraneless organelles including stress granules and nucleoli into a solid phase.
Zhou Lab, Department of Chemistry
ATP is best known as the energy source of cells; for this and other classical roles only micromolar concentrations are required and yet cells maintain millimolars of ATP. One proposed reason for the high levels is that ATP solubilizes protein aggregates by acting as a hydrotrope. Here we investigate a different reason. Our previous study demonstrated that ATP mediates the phase separation of positively charged (or basic) intrinsically disordered proteins (bIDPs). The present study shows that ATP converts various bIDP-nucleic acid aggregates into liquid droplets. ATP displaces the nucleic-acid component and takes millimolar concentrations to outcompete the latter. Under static conditions, it may take many minutes for ATP to complete the displacement; this process is sped up enormously when ATP is supplied in a flow. The fusion of ATP-converted droplets can be slower than the bIDP-ATP droplets, and the slowdown is due to residual nucleic acids inside droplets. In-cell experiments confirm the in vitro effects. Protamine-DNA aggregates are taken up by cells; upon the addition of external ATP, DNA is displaced by ATP from the aggregates and the resulting condensates appear to be liquid. Overall, our findings indicate that the mM ATP levels in cells may be required for keeping IDP-nucleic acid condensates in a liquid state and explain why ATP depletion turns membraneless organelles including stress granules and nucleoli into a solid phase.
Date posted
Jan 13, 2025
Date updated
Jan 13, 2025