Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu. A surprisingly large number of eukaryotic proteins either contain substantial unstructured regions or are entirely unfolded under physiological conditions (1, 2). These "intrinsically disordered proteins" (IDPs) are involved in many crucial cellular processes, such as transcription, translation, and signal transduction; their functional and conformational properties are thus of great interest for a wide range of biological questions. Important advances in understanding the structures of IDPs have been made over the past decade, especially with spectroscopic techniques, e.g., NMR (3, 4), single-molecule fluorescence (5-7), and with atomistic and coarse-grained molecular simulations (8-10). In contrast with the stable folded structures we are familiar with from 50 y of structural biology, IDPs comprise highly heterogeneous and dynamic ensembles of conformations, which either lack stable tertiary structure altogether or fold only on binding their cellular targets (4). Important components of the cellular environment that affect IDPs include not only specific cellular ligands, but also pH and the concentration of salts (11,12). An additional contribution that has been difficult to investigate experimentally comes from the large number of different solutes present in a cell that do not interact with an IDP specifically, but result in an environment that is densely filled with macromolecules and metabolites (12)(13)(14). Given their lack of persistent structure, the conformations of IDPs are expected to be particularly sensitive to the effects of such molecular crowding. Indeed, first experiments indicate that some IDPs gain structure upon crowding (15), whereas others do not (16-18), but may change their dimensions (19)(20)(21). The question of how the conformational distributions of IDPs respond to crowded environments is of particular current interest because IDPs have a vital role in cellular compartments and regions with very high local concentrations of proteins and RNA, such as RNA granules and nuclear pore complexes (22)(23)(24)(25). Howeve...

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The distribution of variation in a quantitative trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. Understanding either distribution is important, because it determines a population's response to natural selection. Unfortunately, existing theory makes conflicting predictions about how selection shapes these distributions, and very little pertinent experimental evidence exists. Here we study a simple genetic system, an evolving RNA enzyme (ribozyme) in which a combination of high throughput genotyping and measurement of a biochemical phenotype allow us to address this question. We show that directional selection, compared to stabilizing selection, increases the genotypic diversity of an evolving ribozyme population. In contrast, it leaves the variance in the phenotypic trait unchanged. AbstractThe distribution of variation in a quantitative trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. Understanding either distribution is important, because it determines a population's response to natural selection. Unfortunately, existing theory makes conflicting predictions about how selection shapes these distributions, and very little pertinent experimental evidence exists. Here we study a simple genetic system, an evolving RNA enzyme (ribozyme) in which a combination of high throughput genotyping and measurement of a biochemical phenotype allow us to address this question. We show that directional selection, compared to stabilizing selection, increases the genotypic diversity of an evolving ribozyme population. In contrast, it leaves the variance in the phenotypic trait unchanged.3