Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.     

Effects of macromolecular crowding on intracellular diffusion from a single particle perspective

Compared to biochemical reactions taking place in relatively well-defined aqueous solutions in vitro, the corresponding reactions happening in vivo occur in extremely complex environments containing only 60-70% water by volume, with the remainder consisting of an undefined array of bio-molecules. In a biological setting, such extremely complex and volume-occupied solution environments are termed 'crowded'. Through a range of intermolecular forces and pseudo-forces, this complex background environment may cause biochemical reactions to behave differently to their in vitro counterparts. In this review, we seek to highlight how the complex background environment of the cell can affect the diffusion of substances within it. Engaging the subject from the perspective of a single particle's motion, we place the focus of our review on two areas: (1) experimental procedures for conducting single particle tracking experiments within cells along with methods for extracting information from these experiments; (2) theoretical factors affecting the translational diffusion of single molecules within crowded two-dimensional membrane and three-dimensional solution environments. We conclude by discussing a number of recent publications relating to intracellular diffusion in light of the reviewed material.     

Protein shape and crowding drive domain formation and curvature in biological membranes

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.     

An early look at macromolecular crowding

Interest in the interaction between proteins and polysaccharides in semidilute systems developed in the late 1950's and early 1960's both in the field of matrix biology and in the construction of new separation techniques. The author gives an account of how his work in the 50's on the characterization of a connective tissue polysaccharide, hyaluronan, led him into studies on polysaccharide networks, sieve effects, molecular exclusion, a theory of gel filtration, solubility of proteins and chemical equilibria in polymer solutions, water and protein homeostatis in vivo, and ordered convectional flow in concentrated polymer systems.     

Protein dynamics and lipid affinity of monomeric,zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from ¹³C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

Protein self-association induced by macromolecular crowding: a quantitative analysis by magnetic relaxation dispersion

In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 510(5)-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions.     

Implications of macromolecular crowding for protein assembly

Recent studies have led to increased appreciation of the influence of excluded volume in solutions of high total macromolecular content ('macromolecular crowding') upon the various classes of reaction that lead to the assembly of proteins and protein complexes. In general, crowding is expected to stabilize native protein structure relative to less compact non-native structures and to favor the formation of functional complexes of native proteins. Under certain pathological conditions, 'overcrowding' may enhance the formation of nonfunctional aggregates of non-native protein (e.g. amyloid and inclusion bodies).     

Self-association of polynucleosome chains by macromolecular crowding

The crowding of macromolecules in the cell nucleus, where their concentration is in the range of 100 mg/ml, is predicted to result in strong entropic forces between them. Here the effects of crowding on polynucleosome chains in vitro were studied to evaluate if these forces could contribute to the packing of chromatin in the nucleus in vivo. Soluble polynucleosomes approximately 20 nucleosomes in length formed fast-sedimenting complexes in the presence of inert, volume-occupying agents poly(ethylene glycol) (PEG) or dextran. This self-association was reversible and consistent with the effect of macromolecular crowding. In the presence of these crowding agents, polynucleosomes formed large assemblies as seen by fluorescence microscopy after labelling DNA with the fluorescent stain DAPI, and formed rods and sheets at a higher concentration of crowding agent. Self-association caused by crowding does not require exogenous cations. Single, approximately 800 nucleosome-long chains prepared in 100 muM Hepes buffer with no added cations, labelled with the fluorescent DNA stain YOYO-1, and spread on a polylysine-coated surface formed compact 3-D clusters in the presence of PEG or dextran. This reversible packing of polynucleosome chains by crowding may help to understand their compact conformations in the nucleus. These results, together with the known collapse of linear polymers in crowded milieux, suggest that entropic forces due to crowding, which have not been considered previously, may be an important factor in the packing of nucleosome chains in the nucleus.     

Molecular crowding and order in photosynthetic membranes

The integrity and maintenance of the photosynthetic apparatus in thylakoid membranes of higher plants requires lateral mobility of their components between stacked grana thylakoids and unstacked stroma lamellae. Computer simulations based on realistic protein densities suggest serious problems for lateral protein and plastoquinone diffusion especially in grana membranes, owing to strong retardation by protein complexes. It has been suggested that three structural features of grana thylakoids ensure efficient lateral transport: the organization of protein complexes into supercomplexes; the arrangement of supercomplexes into structured assemblies, which facilitates diffusion process in crowded membranes; the limitation of the diameter of grana discs to less than approximately 500 nm, which keeps diffusion times short enough to support regulation of light harvesting and repair of photodamaged photosystem II.     

Architectural switches in plant thylakoid membranes

Recent progress in elucidating the structure of higher plants photosynthetic membranes provides a wealth of information. It allows generation of architectural models that reveal well-organized and complex arrangements not only on whole membrane level, but also on the supramolecular level. These arrangements are not static but highly responsive to the environment. Knowledge about the interdependency between dynamic structural features of the photosynthetic machinery and the functionality of energy conversion is central to understanding the plasticity of photosynthesis in an ever-changing environment. This review summarizes the architectural switches that are realized in thylakoid membranes of green plants.     

Effects of macromolecular crowding on protein folding and aggregation

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Protein assembly and crowding simulations

Proteins encounter frequent molecular interactions in biological environments. Computer simulations have become an increasingly important tool in providing mechanistic insights into how such interactions in vivo relate to their biological function. The review here focuses on simulations describing protein assembly and molecular crowding effects as two important aspects that are distinguished mainly by how specific and long-lived protein contacts are. On the topic of crowding, recent simulations have provided new insights into how crowding affects protein folding and stability, modulates enzyme activity, and affects diffusive properties. Recent studies of assembly processes focus on assembly pathways, especially for virus capsids, amyloid aggregation pathways, and the role of multivalent interactions leading to phase separation. Also, discussed are technical challenges in achieving increasingly realistic simulations of interactions in cellular environments.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Diffusion of molecules and macromolecules in thylakoid membranes

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.