The formation of membraneless organelles is vital for the intracellular organization of macromolecules and in regulating many cellular processes. The membraneless organelles are formed by liquid-liquid phase separation (LLPS) mainly constituted of proteins and polynucleotides. The primary factor driving the liquid demixing into two phases is the multivalency of the proteins involved, a general characteristic of intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs). This chapter discusses the role of IDP/IDRs in biomolecular condensate formation and the physical characteristics of these states. Further, the LLPS formation of individual proteins induced by molecular crowding and its relevance to physiological conditions are presented. The studies on the effects of small molecular osmolytes and a hydrotrope, ATP on the phase separation temperature, protein concentration, and reentrant behavior are discussed. The advancements and limitations of the computational methods to predict the phase separation behavior of IDPs, and to analyze the interactions and dynamics of the proteins in condensates are presented. The roles of phase separation in cancer, neurological disorders, and cardiovascular diseases are highlighted.
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