Proteins self-organization is a hallmark of biological systems. Physico-chemical principles governing protein-protein interactions have long been known. However, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a novel strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit an mRNA to the designed micron-scale structures.
The ability to measure the abundance and visualize the localization of proteins across the yeast proteome has stimulated hypotheses on gene function and fueled discoveries. While the classic C’ tagged GFP yeast library has been the only resource for over a decade, the recent development of the SWAT technology has led to the creation of multiple novel yeast libraries where new-generation fluorescent reporters are fused at the N’ and C’ of open reading frames. Efficient access to these data requires a user interface to visualize and compare protein abundance, localization and co-localization across cells, strains, and libraries. YeastRGB (www.yeastRGB.org) was designed to address such a need, through a user-friendly interface that maximizes informative content. It employs a compact display where cells are cropped and tiled together into a ‘cell-grid.’ This representation enables viewing dozens of cells for a particular strain within a display unit, and up to 30 display units can be arrayed on a standard high-definition screen. Additionally, the display unit allows users to control zoom-level and overlay of images acquired using different color channels. Thus, YeastRGB makes comparing abundance and localization efficient, across thousands of cells from different strains and libraries.
The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca, whereas α has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α is preferentially expressed with β in T-tubules of cardiac myocytes, forming αβ heterodimers. We have expressed human αβ, αβ, αβ, and αβ in Pichia pastoris, purified the complexes, and compared their functional properties. αβ and αβ differ significantly from both αβ and αβ in having a higher KK and lower KNa for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K and shift of the EP-EP conformational equilibrium toward EP. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of αβ and αβ over αβ (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K affinity of αβ could allow an acute response to raised ambient K concentrations in physiological conditions and explain the importance of αβ for cardiac muscle contractility. The high sensitivity of αβ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of αβ-selective digoxin derivatives for reducing cardiotoxicity.
The self-organization of proteins into specific assemblies is a hallmark of biological systems. Principles governing protein-protein interactions have long been known. However, principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remains challenging to characterize and understand. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies in living cells. We devise a novel strategy to map high-resolution phase diagrams in vivo , which provide mesoscale self-assembly signatures of our system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how point mutations that change their interaction affinity impact the phase diagram and material state of the assemblies in vivo . Both, the phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Applying our system to interrogate biological mechanisms of self-assembly, we find that co-translational protein binding suffices to recruit an mRNA to the designed micron-scale structures.
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