Biochemical processes inside the cell take place in a complex environment that is highly crowded, heterogeneous, and replete with interfaces. The recently recognized importance of biomolecular condensates in cellular organization has added new elements of complexity to our understanding of chemistry in the cell. Many of these condensates are formed by liquid-liquid phase separation (LLPS) and behave like liquid droplets. Such droplet organelles can be reproduced and studied
in vitro
by using coacervates and have some remarkable features, including regulated assembly, differential partitioning of macromolecules, permeability to small molecules, and a uniquely crowded environment. Here, we review the main principles of biochemical organization in model membraneless compartments. We focus on some promising
in vitro
coacervate model systems that aptly mimic part of the compartmentalized cellular environment. We address the physicochemical characteristics of these liquid phase separated compartments, and their impact on biomolecular chemistry and assembly. These model systems enable a systematic investigation of the role of spatiotemporal organization of biomolecules in controlling biochemical processes in the cell, and they provide crucial insights for the development of functional artificial organelles and cells.
The compartmentalization
of cell-free gene expression systems in
liposomes provides an attractive route to the formation of protocells,
but these models do not capture the physical (crowded) environment
found in living systems. Here, we present a microfluidics-based route
to produce monodisperse liposomes that can shrink almost 3 orders
of magnitude without compromising their stability. We demonstrate
that our strategy is compatible with cell-free gene expression and
show increased protein production rates in crowded liposome protocells.
The formation of
cytomimetic protocells that capture the physicochemical
aspects of living cells is an important goal in bottom-up synthetic
biology. Here, we recreated the crowded cytoplasm in liposome-based
protocells and studied the kinetics of cell-free gene expression in
these crowded containers. We found that diffusion of key components
is affected not only by macromolecular crowding but also by enzymatic
activity in the protocell. Surprisingly, size-dependent diffusion
in crowded conditions yielded two distinct maxima for protein synthesis,
reflecting the differential impact of crowding on transcription and
translation. Our experimental data show, for the first time, that
macromolecular crowding induces a switch from reaction to diffusion
control and that this switch depends on the sizes of the macromolecules
involved. These results highlight the need to control the physical
environment in the design of synthetic cells.
Halogen-bonded complexes with neutral nitroxide radicals as the Lewis base have been investigated in liquid and frozen solutions by multifrequency CW and pulse EPR spectroscopies, including ENDOR and ELDOR-detected NMR (EDNMR) techniques. The non-covalent interaction with iodopentafluorobenzene as halogen-bond donor is shown to affect a variety of EPR parameters of the stable nitroxide radicals. In liquid solution, only bulk effects on the EPR signal, i.e. isotropic
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