Crowded Milieu Research Articles

Sequential Assembly of an Active RNA Polymerase Molecule at the Air−Water Interface

At the heart of understanding cellular processes lies our ability to explore the specific nature of communication between sequential information carrying biopolymers. However, the data extracted from conventional solution phase studies may not reflect the dynamics of communication between recognized partners as they occur in the crowded cellular milieu. We use the principle of immobilization of histidine-tagged biopolymers at a Ni(II)-encoded Langmuir monolayer to study sequence-specific protein-protein interactions in an artificially crowded environment. The advantage of this technique lies in increasing the surface density of one of the interacting partners that allows us to study macromolecular interactions in a controlled crowded environment, but without compromising the speed of the reactions. We have taken advantage of this technique to follow the sequential assembly process of the multiprotein complex Escherichia coli RNA polymerase at the interface and also deciphered the role of one of the proteins, omega (ω), in the assembly pathway. Our reconstitution studies indicate that in the absence of molecular chaperones or other cofactors, omega (ω) plays a decisive role in refolding the largest protein beta prime (β') and its recruitment into the multimeric assembly to reconstitute an active RNA polymerase. It was also observed that the monolayer had the ability to distinguish between sequence-specific and -nonspecific interactions despite the immobilization of one of the biomacromolecules. The technique provides a universal two-dimensional template for studying protein-ligand interactions while mimicking molecular crowding.

Populating the Ambient Space of Texts: The Intimate Graffiti of Doodles. Proposals Toward a Theory

Populating the Ambient Space of Texts: The Intimate Graffiti of Doodles. Proposals Toward a Theory

Accelerated Fibrillation of α-Synuclein Induced by the Combined Action of Macromolecular Crowding and Factors Inducing Partial Folding

To better model the characteristics of crowded intracellular environments, we examined the effect of several factors known to induce partial folding and accelerated fibrillation of alpha-synuclein in dilute solutions, on the fibrillation of this protein in a crowded milieu. We found that low pH, certain metals and pesticides, polyanions, polycations and low concentrations of organic solvents cause a significant acceleration of alpha-synuclein fibrillation in the presence of high concentrations of polyethylene glycol. This suggests that the fibril-promoting effects of factors inducing partial folding of alpha-synuclein and the fibril-stimulating effects of macromolecular crowding are relatively independent and thus might act additively or even synergistically.

Molecular Origami: Protein folding and misfolding in health and disease

Folding within the crowded cellular milieu often requires assistance from molecular chaperones, which prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to protein folding and quality control remain elusive. The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and Prion Disease, is thought to arise upon failure of the cellular protein Quality Control (QC) mechanisms. Our work aims to understand the role of chaperones in folding and in the formation of misfolded protein inclusions in the eukaryotic cytosol. Recently, we identified two intracellular compartments for the sequestration of misfolded cytosolic proteins. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Strikingly, disease‐associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.

Viability in Late Stages of Ex Vivo Erythropoiesis Is Enhanced by Increased Cell Density

Erythropoiesis is one of the body's most productive cell production processes yielding 2×1011 new red cells from hematopoietic stem cells (HSCs) of the bone marrow every day. Intensive research has focused on mimicking this process ex vivo through application of various growth factor combinations or co-culture with stromal cells. To develop a scalable and reproducible system for large scale production of red blood cells we have investigated in vitro erythropoiesis of peripheral blood derived CD34+ cells with primary focus on the impact of the microenvironment on the process. The influence of cultivation conditions on expansion of erythroid progenitor cells and their terminal differentiation to mature red blood cells were studied in stroma-free liquid culture supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO). Peripheral blood derived CD34+ cells were expanded by more than 105 fold over a 3 week period. This degree of expansion has only been achieved previously for CD34+ cells derived from more potent stem cell sources such as cord blood, bone marrow and G-CSF mobilized peripheral blood (Giarratana et al, Nat Biotechnol 2005). The natural environment of human erythropoiesis, the bone marrow, is a very crowded milieu where hematopoietic precursors and other cells are packed in close proximity. Cell crowdedness was found to have significant influences on ex vivo erythropoiesis. Cell density per surface area rather than cell concentration per media volume determined cell expansion during exponential growth where more crowded cells showed reduced overall expansion. In cultures inoculated at 4×105 cells/ml (2.1×105 cells/cm2) increasing cell density per area (i.e. decreasing surface area to volume ratio) 4fold (to 8.4×105 cells/cm2) resulted in 35±12% reduction of total expansion (p<0.05, unpaired Student's t-test). While 4fold increase of cell density in cultures seeded at 1×106 cells/ml (from 5.3×105 cells/cm2 to 2.1×106 cells/cm2) reduced overall expansion by 51±9% (p<0.01). In late stage erythropoiesis, however, when cells had become arrested in G1 and no longer proliferated, cell density was seen to enhance cell viability. Dilution series of late stage erythroblasts showed that although cell viability gradually decreased over a 14 day cultivation period the decreasing rate was lower in cells cultivated at higher density as shown in the Figure. Enhanced viability in crowded culture conditions could reflect the cells' dependency on direct cell-cell interactions as found in the marrow environment. Cultures grown to high cell densities of 2–3×106 cells/cm2 showed higher maturation efficiency than previously obtained in this cultivation set-up with more than 80% of cells being CD71-/GpA+. Enucleation yields of up to 45% were achieved indicating a significant amount of terminal maturation to red blood cells. Efficient maturation and particularly enucleation have in many cases been found to be dependent on or improved by interactions with feeder cells or macrophages (Fujimi et al, Int J Hematol 2008). Keeping erythroid cells at high densities during late stages of erythropoiesis possibly helps to mimic their in vivo environment, thus allowing for better survival and efficient terminal maturation without the need for co-culture with other cells. [Display omitted]

Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies

Folding within the crowded cellular milieu often requires assistance from molecular chaperones that prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to folding the proteome remains elusive. We here demonstrate that the eukaryotic chaperonin TRiC/CCT (TCP1-Ring Complex or Chaperonin Containing TCP1) has broad binding specificity in vitro similar to the prokaryotic chaperonin GroEL. However, in vivo TRiC substrate selection is not based solely on intrinsic determinants; instead, specificity is dictated by factors present during protein biogenesis. The identification of cellular substrates revealed that TRiC interacts with folding intermediates of a subset of structurally and functionally diverse polypeptides. Bioinformatics analysis revealed an enrichment in multidomain proteins and regions of beta strand propensity that are predicted to be slow-folding and aggregation-prone. Thus, TRiC may have evolved to protect complex protein topologies within its central cavity during biosynthesis and folding.

Integrated biophotonics approach for noninvasive and multiscale studies of biomolecular and cellular biophysics

In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, noninvasive, multiscale experimental approach. Here, we develop a multimodal fluorescence microspectroscopy system, integrated on a single platform, to gain information about molecular interactions and their dynamics with high spatio-temporal resolution. To demonstrate the versatility of our experimental approach, we use rhodamine 123-labeled mitochondria in breast cancer cells (Hs578T), verified using differential interference contrast (DIC) and fluorescence (confocal and two-photon) microscopy, as a model system. We develop an assay to convert fluorescence intensity to actual concentrations in intact, individual living cells, which contrasts with conventional biochemical techniques that require cell lysates. In this assay, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify the fluorescence quantum yield variations found within individual cells. Functionally driven changes in cell environment, molecular conformation, and rotational diffusion are investigated using fluorescence polarization anisotropy imaging. Moreover, we quantify translational diffusion and chemical kinetics of large molecular assemblies using fluorescence correlation spectroscopy. Our integrated approach can be applied to a wide range of molecular and cellular processes, such as receptor-mediated signaling and metabolic activation.

Effects of Macromolecular Crowding on Glycoprotein Processing Enzymes

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.

Albumin is a redox‐active crowding agent that promotes oxidative folding of cysteine‐rich peptides

Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To the best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxin-based protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER-resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors.

Nucleoid organization and the maintenance of DNA integrity in E. coli, B. subtilis and D. radiodurans

For enzymatic activities to be effectively carried out, basic prerequisites must be met. Many enzymatic tasks require continuous consumption and dissipation of energy, sometimes in massive amounts. Some activities, such as DNA replication, transcription, and repair through homologous recombination rely upon templates that provide the information required for these transactions. Yet, circumstances where intracellular energy pools are severely depleted, or where intact templates are not available, frequently occur. Moreover, the fact that in order to reach their targets, enzymes must cope with an extremely crowded and viscous cellular milieu that drastically slows down their diffusion is often neglected. These impediments are particularly evident under stress conditions such as prolonged starvation or continuous exposure to DNA-damaging agents. Here we survey recent studies, which imply that when enzymatically-mediated DNA repair pathways are hindered, alternative strategies are deployed, whose common denominator is the reorganization of bacterial nucleoids into morphologies that promote DNA repair and protection.

The Nitty-Gritty of Protein Interactions

In this issue of Structure , Cho et al. (2005) provide a detailed structural analysis of the molecular mechanism of affinity maturation for the murine T cell receptor Vβ domain. Information from nine X-ray structures of combinations along the maturation pathway and comprehensive thermodynamics reveal the fine details of high-affinity binding.

Oligomeric Hsp33 with Enhanced Chaperone Activity: GEL FILTRATION, CROSS-LINKING, AND SMALL ANGLE X-RAY SCATTERING (SAXS) ANALYSIS

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.

Effect of Crowding on Protein–Protein Association Rates: Fundamental Differences between Low and High Mass Crowding Agents

Physiological media constitutes a crowded environment that serves as the field of action for protein–protein interaction in vivo. Measuring protein–protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein–protein association and its rate constants ( k on) of the β-lactamase (TEM)–β-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0–40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k on, but not k off values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k on depends inversely on the solution viscosity. In high mass polymer solutions k on changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length ( L) and its persistence length ( L p). Polymers that are long enough to form a flexible coil ( L/ L p>2) behave as high molecular mass polymers and those who are unable to do so ( L/ L p<2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k on of wt-TEM and three BLIP analogs with up to 100-fold increased values of k on due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment.

Protein folding and misfolding.

The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.

Accelerated α-synuclein fibrillation in crowded milieu

Parkinson’s disease is the second most common age-related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α-synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/l. To address this discrepancy, the effect of molecular crowding on α-synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethylene glycols) dramatically accelerated α-synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume.