The Study Of Stability,Dynamics And Conformation Of Biomacromolecules In Membraneless Organelles By NMR Spectroscopy

Membraneless organelles are aggregates of proteins and nucleic acids with liquid properties in the cell.These subcellular compartments are formed by liquid-liquid phase separation of proteins or nucleic acids.Protein phase separation is involved in many important physiological processes of cells and has been extensively studied.The study found that the intracellular activities involved in phase separation include the formation of membraneless organelles,supramolecular assembly,signal transduction,gene activation,and cytoskeleton.In addition,phase separation is closely related to certain diseases such as neurodegenerative diseases.At present,the research methods of phase separation mainly focus on molecular biology,in vitro recombination and microscopic imaging,and the use of nuclear magnetic resonance to study protein phase separation is relatively few.It has been reported that ATP-dependent RNA helicase DDX4 undergoes phase separation in vitro and in cells to form small droplets,and external conditions such as temperature and salt concentration can regulate the phase separation of DDX4.The disordered N-terminal of DDX4N1(DDX4N1)composed of 236 amino acid residues can undergo phase separation in vitro to form small droplets with a diameter of micron.The function of these membraneless organelles is considered as a "filter" or"concentrator",and can selectively recruit certain biological macromolecules to these small droplets.The study found that DDX4N1 can recruit single-stranded nucleic acids into the droplets formed by phase separation and stabilize single-stranded nucleic acids,but exclude double-stranded nucleic acids;for proteins,these droplets exhibit a wide range of absorption or exclusion capabilities,and those proteins that can be recruited can act as importers for otherwise-excluded nucleic acids.Whether the structure,function and dynamics of the recruited biological macromolecules will change,or the effect of the different recruited biological macromolecules on these small droplets is now unclear.We used nuclear magnetic resonance and other biophysical methods to determine the phase separation conditions of DDX4N1 in solution and selected a protein labeling method suitable for NMR studies.We selected two types of biomacromolecules,SH3 and G-quadruplexes,and studied their structure,stability,and kinetics when they are enveloped into the droplets formed by DDX4N1 phase separation.The results show that the SH3 and G-quadruplexes can be wrapped into the droplets formed by DDX4N1 phase separation,but the SH3 and G-quadruplexes have different effects on the phase separation of DDX4N1.SH3 will inhibit the phase separation of DDX4N1,while G-quadruplexes promotes the phase separation of DDX4N1.The interactions of SH3 and G-quadruplexes with DDX4N1 are also different.The interaction of SH3 and DDX4N1 is very weak,while the interaction of G-quadruplexes and DDX4N1 is stronger.We measured the partition coefficient of different concentration of SH3 in the 200?M DDX4N1 droplets.The results show that when the concentration of DDX4N1 is unchanged,the efficiency of DDX4N1 to wrap SH3 is different when the initial concentration of SH3 is different.When the concentration of SH3 is close to that of DDX4N1,SH3 enters DDX4N1 droplets most efficiently,and the partition coefficients of SH3 in the droplets is largest.We centrifuged to separate the droplets of DDX4N1 wrapped SH3,and obtained the SH3-wrapped DDX4N1 condensate.The study found that the proportion of the two states of SH3 changed significantly in the DDX4N1 condensate,that is the proportion of the fold state was greatly reduced,and the proportion of the unfold state was greatly increased.The results of thermodynamic parameter show that the stability of SH3 is reduced in DDX4N1 condensate,which is consistent with the proportion of its two states.In addition,the kinetic parameters also changed significantly,the T1 value increased significantly,while the T2 value decreased significantly,and the exchange rate of its two states also changed.By simulating the environment of DDX4N1 condensate in Ficoll 70,we concluded that the change of the exchange rate of SH3 is not caused by the crowded environment or viscosity increase,the weak interaction between DDX4N1 and SH3 is an important factor affecting the exchange rate of the two states.We observed that when the biological macromolecules were recruited into phase-separated droplets,the balance between folded and unfolded conformations changed,suggesting that the weak interactions between macromolecules in organisms may participate in important molecular regulation.Our results provide experimental data for studying the phase separation of cells,and give insights to the understanding of the biological function of phase separation.