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Structural engineering of molecules for condensation is an emerging technique within synthetic biology. Liquid-liquid phase separation of biomolecules leading to condensation is a central step in the assembly of biological materials into their functional forms. Intracellular condensates can also function within cells in a regulatory manner to facilitate reaction pathways and to compartmentalize interactions. We need to develop a strong understanding of how to design molecules for condensates and how their in vivo- in vitro properties are related. The spider silk protein NT2RepCT undergoes condensation during its fiber-forming process. Using parallel in vivo and in vitro characterization, in this study, we mapped the effects of intracellular conditions for NT2RepCT and its several structural variants. We found that intracellular conditions may suppress to some extent condensation whereas molecular crowding affects both condensate properties and their formation. Intracellular characterization of protein condensation allowed experiments on pH effects and solubilization to be performed within yeast cells. The growth of intracellular NT2RepCT condensates was restricted, and Ostwald ripening was not observed in yeast cells, in contrast to earlier observations in E. coli . Our results lead the way to using intracellular condensation to screen for properties of molecular assembly. For characterizing different structural variants, intracellular functional characterization can eliminate the need for time-consuming batch purification and in vitro condensation. Therefore, we suggest that the in vivo - in vitro understanding will become useful in, e.g., high-throughput screening for molecular functions and in strategies for designing tunable intracellular condensates.

Abstract: Recently obtained results on gas–polymer interactions and associated molecular organizations are reported. As a matter of fact, they are of interest for several applications from petroleum industry to nano-science and polymer processing. They constitute an incursion into the thermodynamics and kinetics of solid polymer/gas interactions and liquid polymer/gas interactions as well as solid to liquid (polymer) transitions. Experimental and theoretical studies have been conducted to better understand the competition between the effects of solubility/plasticization and of hydrostatic pressure of a gas in the polymer-rich phase and also the importance of the interface in block copolymers. A relation characterizing the global isobaric thermal expansion of thermoplastic semi-crystalline polymers saturated in gas has been developed. In addition, it has been shown that the control of the supercritical conditions of the gas enabled to successfully monitor the size of nano-structures appearing by self-assembling in di-block copolymers. The overall kinetics of the liquid to solid transition, that is crystallization, of a semi-crystalline polymer has been described within the framework of Avrami general assumptions. A system of differential equations has been derived for quiescent crystallization and extended to flow-induced crystallization which can be used to predict the formation of microstructures. [Copyright &y& Elsevier]