Reconstitution Experiments of Biomolecular Condensates Intrinsically Disordered Proteins

Intrinsically disordered protein regions (IDRs) typically drive phase separation. In addition, biomolecular condensates contain many proteins with IDRs suggesting a role in protein recruitment and possibly in shaping the properties of the condensate. Different biomolecular condensates have different sequence-specific IDRs, meaning that IDRs can encode phase behavior in many different ways, and that IDRs can confer phase separation specificity and control which proteins co-phase separate. Many types of potential molecular interactions have been shown to help drive phase separation of IDRs, including hydrophobic, electrostatic, pi-pi, cation-pi, and hydrogen bonding interactions. Given the critical role of phase separation in many cellular processes, phase separation dysfunction can lead to debilitating diseases, so it is important to understand the interactions and sequence properties behind phase behavior.

Fig. 1. Conceptualizing liquid-liquid phase separation of IDRs. (Borcherds W, et al., 2021)

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We provide the simple but powerful stickers-and-spacers framework to conceptually understand the drivers of phase separation in IDRs and which elements of the sequence are most important for their phase behavior. Here, we focus on recombination experiments to analyze protein scaffolds in biomolecular condensates (which use structural domains to mediate the protein-protein or protein-RNA interactions that drive phase separation) by purifying and mixing proteins in vitro, or by using transgenes expressed in cells and examined for condensation formation by microscopy or centrifugation.

Our reconstitution experiments not only identify the range of molecular interactions that promote phase separation, but also detect in vivo biomolecular condensates to determine the binding interactions that drive phase separation in cells. CD BioSciences offers comprehensive reconstitution experiments of biomolecular condensates intrinsically disordered proteins to analyze the binding interactions that support the assembly of P-bodies, germ granules, stress granules, and other cohesions formed in the cytoplasm.

• Identification of Proteins Necessary for Phase Separation
  Most cellular cohesions contain dozens of proteins, and we can identify proteins that are essential for phase separation in vivo ("scaffolds"), including but not limited to:

• P-bodies
• Germ granules
• Transport granules
• Whi3 granules
• TIS granules
• Pyrenoid
• Stress-induced cytoplasmic condensates
• Pathological condensates

• Identification of Basic Structural Domains
  Once the scaffolds are identified, we perform structural and functional studies on these proteins to determine the basic structural domains. For mutant versions, we assay in vivo using methods that minimize overexpression, which can confound results by artificially increasing intracellular concentrations or distorting the scaffold-to-client ratio.

Advances in genetic engineering and proteomics have made it possible to systematically interrogate IDR sequences in vivo. CD BioSciences offers recombinant experiments using purified or overexpressed proteins to identify structural domains of proteins that have the potential to drive phase separation. We have successfully identified candidate polyvalent scaffolds for several natural biomolecular condensates. Understanding how IDR facilitates the assembly of biomolecular condensates can provide insights into the mechanisms of cellular control of dynamics, material properties, and ultimately function. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.