Low amounts of heavy water increase the phase separation propensity of a fragment of the androgen receptor activation domain

Abstract

The phase equilibria of intrinsically disordered proteins are exquisitely sensitive to changes in solution conditions and this can be used to investigate the driving forces of phase separation in vitro as well as the biological roles of phase transitions in live cells. Here we investigate how using D2O as co‐solvent in an aqueous buffer changes the phase equilibrium of a fragment of the activation domain of the androgen receptor, a transcription factor that plays a role in the development of the male phenotype and is a therapeutic target for castration resistant prostate cancer. We show how replacing even small fractions of H2O with D2O increases the propensity of this fragment to undergo liquid–liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation. Our results indicate that it is necessary to take this effect into consideration when studying phase separation phenomena with biophysical methods that require using D2O as a co‐solvent. In addition, they suggest that additions of D2O may be used to enhance phase separation phenomena in cells, facilitating their observation.

Dynamic phase separation of the androgen receptor and its coactivators key to regulate gene expression

“…Within this context, recent discoveries regarding phase separation of AR are of high interest. It has been demonstrated that the intrinsically disordered N-terminal domain of AR forms liquid-like droplets in vitro at ∼100 μM ( 17 ), and the isolated Tau 5 region within this NTD at ∼500 μM ( 18 ). Moreover, the isolated DBD was shown to undergo phase separation in presence of RNA (poly U) ( 19 ).…”

Topological dynamics of an intrinsically disordered N‐terminal domain of the human androgen receptor

“…Some important steps have been taken including insights from single‐molecule fluorescence resonance energy transfer (smFRET), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR) 13 . However, none have been applied successfully to resolve the structure of the full‐length AR‐NTD due to technical difficulties 14–16 …”

“…13 However, none have been applied successfully to resolve the structure of the fulllength AR-NTD due to technical difficulties. [14][15][16] The intrinsic disorder poses a challenge not only for experimental analysis of the conformation but also for computational modeling of the chain due to the size of the conformation space and lack of stable folds. For example, the state-of-the art artificial intelligence-based prediction approaches fail to identify the conformation of AR-NTD.…”

Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy

“…Another fluorinated molecule (TFT) in a coaxial insert acts as an external reference 39 , and enables observation of magnetic field inhomogeneity emerging as a result of macroscopic layer separation, without the complication of the reference solution itself participating in LLPS. The coaxial insert is also crucial for housing deuterated solvent for field-frequency lock required by the NMR spectrometer, thereby avoiding the addition of 2 H 2 O directly to the protein solution, which is known to alter the LLPS propensity of proteins 33 , 40 , 41 . Despite the presence of two distinct phases emerging during the initial microscopic phase separation, magnetic field homogeneity, as judged by TFT signal lineshape, is initially remarkably unperturbed.…”