The differential response of proteins to macromolecular crowding

The habitat in which proteins exert their function contains up to 400 g/L of macromolecules, most of which are proteins. The repercussions of this dense environment on protein behavior are often overlooked or addressed using synthetic agents such as poly(ethylene glycol), whose ability to mimic protein crowders has not been demonstrated. Here we performed a comprehensive atomistic molecular dynamic analysis of the effect of protein crowders on the structure and dynamics of three proteins, namely an intrinsically disordered protein (ACTR), a molten globule conformation (NCBD), and a one-fold structure (IRF-3) protein. We found that crowding does not stabilize the native compact structure, and, in fact, often prevents structural collapse. Poly(ethylene glycol) PEG500 failed to reproduce many aspects of the physiologically-relevant protein crowders, thus indicating its unsuitability to mimic the cell interior. Instead, the impact of protein crowding on the structure and dynamics of a protein depends on its degree of disorder and results from two competing effects: the excluded volume, which favors compact states, and quinary interactions, which favor extended conformers. Such a viscous environment slows down protein flexibility and restricts the conformational landscape, often biasing it towards bioactive conformations but hindering biologically relevant protein-protein contacts. Overall, the protein crowders used here act as unspecific chaperons that modulate the protein conformational space, thus having relevant consequences for disordered proteins

Anomalous transport in the crowded world of biological cells

A ubiquitous observation in cell biology is that diffusion of macromolecules and organelles is anomalous, and a description simply based on the conventional diffusion equation with diffusion constants measured in dilute solution fails. This is commonly attributed to macromolecular crowding in the interior of cells and in cellular membranes, summarising their densely packed and heterogeneous structures. The most familiar phenomenon is a power-law increase of the MSD, but there are other manifestations like strongly reduced and time-dependent diffusion coefficients, persistent correlations, non-gaussian distributions of the displacements, heterogeneous diffusion, and immobile particles. After a general introduction to the statistical description of slow, anomalous transport, we summarise some widely used theoretical models: gaussian models like FBM and Langevin equations for visco-elastic media, the CTRW model, and the Lorentz model describing obstructed transport in a heterogeneous environment. Emphasis is put on the spatio-temporal properties of the transport in terms of 2-point correlation functions, dynamic scaling behaviour, and how the models are distinguished by their propagators even for identical MSDs. Then, we review the theory underlying common experimental techniques in the presence of anomalous transport: single-particle tracking, FCS, and FRAP. We report on the large body of recent experimental evidence for anomalous transport in crowded biological media: in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where model systems mimic physiological crowding conditions. Finally, computer simulations play an important role in testing the theoretical models and corroborating the experimental findings. The review is completed by a synthesis of the theoretical and experimental progress identifying open questions for future investigation.Comment: review article, to appear in Rep. Prog. Phy

The macroscopic effects of microscopic heterogeneity

Over the past decade, advances in super-resolution microscopy and particle-based modeling have driven an intense interest in investigating spatial heterogeneity at the level of single molecules in cells. Remarkably, it is becoming clear that spatiotemporal correlations between just a few molecules can have profound effects on the signaling behavior of the entire cell. While such correlations are often explicitly imposed by molecular structures such as rafts, clusters, or scaffolds, they also arise intrinsically, due strictly to the small numbers of molecules involved, the finite speed of diffusion, and the effects of macromolecular crowding. In this chapter we review examples of both explicitly imposed and intrinsic correlations, focusing on the mechanisms by which microscopic heterogeneity is amplified to macroscopic effect.Comment: 20 pages, 5 figures. To appear in Advances in Chemical Physic

Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3d individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion- aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.Comment: PLoS Computational Biology (2013

A Multi-Layered Study on Harmonic Oscillations in Mammalian Genomics and Proteomics

Cellular, organ, and whole animal physiology show temporal variation predominantly featuring 24-h (circadian) periodicity. Time-course mRNA gene expression profiling in mouse liver showed two subsets of genes oscillating at the second (12-h) and third (8-h) harmonic of the prime (24-h) frequency. The aim of our study was to identify specific genomic, proteomic, and functional properties of ultradian and circadian subsets. We found hallmarks of the three oscillating gene subsets, including different (i) functional annotation, (ii) proteomic and electrochemical features, and (iii) transcription factor binding motifs in upstream regions of 8-h and 12-h oscillating genes that seemingly allow the link of the ultradian gene sets to a known circadian network. Our multifaceted bioinformatics analysis of circadian and ultradian genes suggests that the different rhythmicity of gene expression impacts physiological outcomes and may be related to transcriptional, translational and post-translational dynamics, as well as to phylogenetic and evolutionary components

Nanoporous silica-based protocells at multiple scales for designs of life and nanomedicine.

Various protocell models have been constructed de novo with the bottom-up approach. Here we describe a silica-based protocell composed of a nanoporous amorphous silica core encapsulated within a lipid bilayer built by self-assembly that provides for independent definition of cell interior and the surface membrane. In this review, we will first describe the essential features of this architecture and then summarize the current development of silica-based protocells at both micro- and nanoscale with diverse functionalities. As the structure of the silica is relatively static, silica-core protocells do not have the ability to change shape, but their interior structure provides a highly crowded and, in some cases, authentic scaffold upon which biomolecular components and systems could be reconstituted. In basic research, the larger protocells based on precise silica replicas of cells could be developed into geometrically realistic bioreactor platforms to enable cellular functions like coupled biochemical reactions, while in translational research smaller protocells based on mesoporous silica nanoparticles are being developed for targeted nanomedicine. Ultimately we see two different motivations for protocell research and development: (1) to emulate life in order to understand it; and (2) to use biomimicry to engineer desired cellular interactions