Question

What does the title phrase ‘overcrowded milieu’ mean in the context of intrinsically disordered proteins, proteinaceous membrane-less organelles, and everything else constituting the cytoplasm? How do you think IDPs and PMLOs fit into the overall cytoplasmic content and at what ratio?

Answer 1

Although the cellular interior is crowded with various biological macromolecules, the distribution of these macromolecules is highly inhomogeneous. Eukaryotic cells contain numerous proteinaceous membrane-less organelles (PMLOs), which are condensed liquid droplets formed as a result of the reversible and highly controlled liquid-liquid phase transitions. The interior of these cellular bodies represents an overcrowded milieu since their protein concentrations are noticeably higher than those of the crowded cytoplasm and nucleoplasm. PMLOs are different in size, shape, and composition, and almost invariantly contain intrinsically disordered proteins.

Effects of macromolecular crowding on structural and functional properties of ordered proteins, their folding, interactability, and aggregation are well documented. Much less is known about how macromolecular crowding might affect the structural and functional behavior of intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDPRs). To fill this gap, this review represents a systematic analysis of the available literature data on the behavior of IDPs/IDPRs in a crowded environment. Although it was hypothesized that, due to the excluded-volume effects present in crowded environments, IDPs/IDPRs would invariantly fold in the presence of high concentrations of crowding agents or in the crowded cellular environment, accumulated data indicate that, based on their response to the presence of crowders, IDPs/IDPRs can be grouped into three major categories, foldable, non-foldable, and unfoldable. This is because the natural cellular environment is not simply characterized by the presence of a high concentration of “inert” macromolecules, but represents an active milieu, components of which are engaged in direct physical interactions and soft interactions with target proteins. Some of these interactions with cellular components can cause (local) unfolding of query proteins. In other words, since crowding can cause both folding and unfolding of an IDP or its regions, the outputs of the placing of a query protein to the crowded environment would depend on the balance between these two processes. As a result, and because of the Spatio-temporal heterogeneity in the structural organization of IDPs, macromolecular crowding can differently affect structures of different IDPs. Recent studies indicate that some IDPs are able to undergo liquid–liquid-phase transitions leading to the formation of various proteinaceous membrane-less organelles (PMLOs). Although interiors of such PMLOs are self-crowded, being characterized by locally increased concentrations of phase-separating IDPs, these IDPs are minimally foldable or even non-foldable at all (at least within the physiologically safe time-frame of normal PMLO existence).