Compartmentalization of biochemical processes is essential for cell function. Although membrane‐bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid‐like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non‐equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function.
The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.
Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid‐liquid phase separation. The so‐called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein “structure‐function paradigm,” as their functions do not necessarily involve well‐defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single‐molecule fluorescence methods can overcome this ensemble‐average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single‐molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in‐depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel
Proper cell function requires specific physical and chemical interactions to take place among high concentrations of macromolecules, small molecules, and ions. One important means by which this specificity is achieved is by compartmentalization of biochemistry. Recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid‐like membraneless organelles (MLOs). In their Minireview on page 5600 ff., E. Bentley, B. Frey and A. Deniz discuss the key conceptual and mechanistic underpinnings of this phenomenon, including weak noncovalent interactions and polymer physics principles, biological examples and active matter.
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