Elucidating the liquid structure within biomolecular condensates.

Abstract

Biomolecular condensates are liquid-like membrane-less organelles where essential biochemistry occurs, including ribosome and spliceosome biogenesis. Many studies posit that to carry out biochemical processes, the condensate preferentially enriches or excludes specific molecules. Unlike gases, condensates are liquid-like phases and thus can have locally correlated structure. Such liquid structure may facilitate long-range conformational changes (i.e., allostery) between biomolecules, spatiotemporally coordinating interactions that direct binding, processing, and folding of biomolecules. Measurement of structure is classically obtained through diffraction-based methods, which is not conducive to condensates in cells. We instead turned to a different approach using live cell fluorescent microscopy to extract the strength driving molecules into condensates, quantified by their partitioning (i.e., free energy of transfer). To accomplish this, we employed a construct with two domains separated by a variable-sized linker. We surmised that its partitioning would depend on the exact length of the linker. For example, if the linker is too short or too long the transfer free energy would weaken as the domains are infrequently capable of “reaching” their preferred interaction partners within the condensate. Based on this intuition and other thermodynamic considerations, we converted these free energies into a measure of the degree of correlation between the two domains, known as the pair correlation function. Employing this scheme on common condensate driver proteins (e.g., NPM1 and G3BP1) allowed us to elucidate general principles underlying preferred molecular arrangements within biological condensates. Furthermore, by perturbing nucleolar composition and identifying how the degree of its structure is impacted, we gain insights into the connection between nucleolar form and ribosome biogenesis. Overall, this novel methodology opens the door to understanding the liquid structure of condensates, how they mechanistically facilitate function, and how they may change during development, aging, and disease.