Periodic Reporting for period 1 - BiophInLLPSInt (Biophysical investigation of the liquid-liquid phase separation solvent interface.)
Reporting period: 2023-01-01 to 2024-12-31
Liquid-liquid phase separation (LLPS) is a biophysical phenomenon through which biomolecules condense and accumulate locally. This effect has been linked to a high increase of activity for enzymatic systems in vitro. To form LLPS, biomolecules interact through a mix of specific and/or non-specific interactions, often transient in nature and involving multivalency. In vitro models of biological LLPS often rely on molecular crowders to induce condensation.
Akin to many other phase-separating biological systems, several elements of the DNA double-strand break repair pathway of non-homologous end-joining (NHEJ) have been shown in our lab to undergo phase separation in vitro in presence of crowding agents. The NHEJ process involves the sequential collective action of numerous proteins to successively tether the severed DNA ends, form synapsis, and ligate the DNA. Of interest to our project are the scaffolding protein homodimers XRCC4 and XLF, and the DNA ligase IV. These proteins consist of both folded and very dynamic intrinsically disordered domains, and are involved in in-cis and in-trans interactions. These three proteins are together already sufficient to form condensates in vitro and in presence of crowding agents – which strongly increases ligation activity in presence of blunt-end linear DNA.
However, there is a conundrum: although these three components can condense at low concentrations, slightly below one micromolar, they are present in sub-LLPS concentrations in vivo. How do they get recruited to the NHEJ complex during DNA-repair? How do components issued from the dilute phase enter condensates? What happens at the surface of the condensates?
We aimed to use the molecular insider’s view provided by NMR and especially Relaxation and high-resolution relaxometry (HRR) to study the behaviour of an NHEJ component interacting with condensates at atomic resolution.
Akin to many other phase-separating biological systems, several elements of the DNA double-strand break repair pathway of non-homologous end-joining (NHEJ) have been shown in our lab to undergo phase separation in vitro in presence of crowding agents. The NHEJ process involves the sequential collective action of numerous proteins to successively tether the severed DNA ends, form synapsis, and ligate the DNA. Of interest to our project are the scaffolding protein homodimers XRCC4 and XLF, and the DNA ligase IV. These proteins consist of both folded and very dynamic intrinsically disordered domains, and are involved in in-cis and in-trans interactions. These three proteins are together already sufficient to form condensates in vitro and in presence of crowding agents – which strongly increases ligation activity in presence of blunt-end linear DNA.
However, there is a conundrum: although these three components can condense at low concentrations, slightly below one micromolar, they are present in sub-LLPS concentrations in vivo. How do they get recruited to the NHEJ complex during DNA-repair? How do components issued from the dilute phase enter condensates? What happens at the surface of the condensates?
We aimed to use the molecular insider’s view provided by NMR and especially Relaxation and high-resolution relaxometry (HRR) to study the behaviour of an NHEJ component interacting with condensates at atomic resolution.
The NHEJ components and one single LLPS interacting domain (SLID) have been produced recombinantly and are biochemically active. In the process of characterising the dynamics of our SLID model, I acquired full relaxation and high-resolution relaxometry of our SLID in presence of two different crowding agents, which is the environment of the dilute phase in phase-separated systems. Understanding dynamics of our disordered protein in a mimic of the dilute phase is a prerequisite to the investigation of the interface between the dilute phase and the dense phase. The dynamics of our SLID are strongly affected by the presence of crowders, and by the nature of the crowder itself. These unprecedented datasets require a refinement of models of motion to be fully analyzed. This analysis is still underway. We have implemented some new models in the MINOTAUR software.
Preliminary works on the quantification of the SLID recruitment into the dense phase, and on proper sample preparation of SLID-LLPS mix for NMR have been undertaken using fluorescence spectroscopy and microscopy.
To further characterise the effect of crowders – owing to their central role to the LLPS process – high-resolution relaxometry datasets have been measured on various crowder preparations, varying crowder type and concentration ass well as the presence of dissolved oxygen. A dedicated fitting software has been written and we are currently analyzing this large dataset.
Preliminary works on the quantification of the SLID recruitment into the dense phase, and on proper sample preparation of SLID-LLPS mix for NMR have been undertaken using fluorescence spectroscopy and microscopy.
To further characterise the effect of crowders – owing to their central role to the LLPS process – high-resolution relaxometry datasets have been measured on various crowder preparations, varying crowder type and concentration ass well as the presence of dissolved oxygen. A dedicated fitting software has been written and we are currently analyzing this large dataset.
Although some challenges survive, the dynamics of folded proteins on pico and nanosecond timescales has been investigated for over three decades by NMR relaxation and is reasonably well understood. On the other hand, understanding motions of disordered proteins on nano- to microsecond timescales has been an active domain of research for a few expert groups in the past decade. Using high-resolution relaxometry has great potential unraveling protein dynamics at nanosecond to microsecond timescales, and the results of our SLID dynamics will increase our understanding of the dynamics of intrinsically disordered proteins.
Molecular crowders are often used as a surrogate to mimic the cellular environment, and are sometimes used interchangeably. There is still discussion on the nature of the crowding effect on proteins. Currently, there is only a single investigation on the effect of crowders on the dynamics of disordered proteins. In particular, HRR is uniquely suited to uncover changes in the distribution of slow nanosecond motions. Our analysis indicates that slow motional modes (slower than 10 ns), may be uncovered by our approach. The understanding from our SLID relaxation with crowders, as well as water and crowder relaxometry will bring useful information on the nature of crowding and its effect on the motions of disordered proteins.
Molecular crowders are often used as a surrogate to mimic the cellular environment, and are sometimes used interchangeably. There is still discussion on the nature of the crowding effect on proteins. Currently, there is only a single investigation on the effect of crowders on the dynamics of disordered proteins. In particular, HRR is uniquely suited to uncover changes in the distribution of slow nanosecond motions. Our analysis indicates that slow motional modes (slower than 10 ns), may be uncovered by our approach. The understanding from our SLID relaxation with crowders, as well as water and crowder relaxometry will bring useful information on the nature of crowding and its effect on the motions of disordered proteins.