Artykuły w czasopismach: „Macromolecular crowding agents”

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De Pieri, Andrea, Shubhasmin Rana, Stefanie Korntner i Dimitrios I. Zeugolis. "Seaweed polysaccharides as macromolecular crowding agents". International Journal of Biological Macromolecules 164 (grudzień 2020): 434–46. http://dx.doi.org/10.1016/j.ijbiomac.2020.07.087.

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Lee, Hong Bok, Anh Cong, Hannah Leopold, Megan Currie, Arnold J. Boersma, Erin D. Sheets i Ahmed A. Heikal. "Rotational and translational diffusion of size-dependent fluorescent probes in homogeneous and heterogeneous environments". Physical Chemistry Chemical Physics 20, nr 37 (2018): 24045–57. http://dx.doi.org/10.1039/c8cp03873b.

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Stepanenko, Olesya V., Olga V. Stepanenko, Irina M. Kuznetsova i Konstantin K. Turoverov. "The unfolding of iRFP713 in a crowded milieu". PeerJ 7 (8.04.2019): e6707. http://dx.doi.org/10.7717/peerj.6707.

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The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.

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del Álamo, Marta, Germán Rivas i Mauricio G. Mateu. "Effect of Macromolecular Crowding Agents on Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro". Journal of Virology 79, nr 22 (15.11.2005): 14271–81. http://dx.doi.org/10.1128/jvi.79.22.14271-14281.2005.

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ABSTRACT Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.

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Gnutt, David, i Simon Ebbinghaus. "The macromolecular crowding effect – from in vitro into the cell". Biological Chemistry 397, nr 1 (1.01.2016): 37–44. http://dx.doi.org/10.1515/hsz-2015-0161.

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Abstract The influence of the cellular milieu, a complex and crowded solvent, is often neglected when biomolecular structure and function are studied in vitro. To mimic the cellular environment, crowding effects are commonly induced in vitro using artificial crowding agents like Ficoll or dextran. However, it is unclear if such effects are also observed in cellulo. Diverging results on protein stability in living cells point out the need for new quantitative methods to investigate the contributions of excluded volume and nonspecific interactions to the cellular crowding effect. We show how new crowding sensitive probes may be utilized to directly investigate crowding effects in living cells. Moreover, we discuss processes where crowding effects could play a crucial role in molecular cell biology.

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Vweza, Alick-O., Chul-Gyu Song i Kil-To Chong. "Liquid–Liquid Phase Separation in the Presence of Macromolecular Crowding and State-Dependent Kinetics". International Journal of Molecular Sciences 22, nr 13 (22.06.2021): 6675. http://dx.doi.org/10.3390/ijms22136675.

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Biomolecular condensates formed via liquid–liquid phase separation (LLPS) are increasingly being shown to play major roles in cellular self-organization dynamics in health and disease. It is well established that macromolecular crowding has a profound impact on protein interactions, particularly those that lead to LLPS. Although synthetic crowding agents are used during in vitro LLPS experiments, they are considerably different from the highly crowded nucleo-/cytoplasm and the effects of in vivo crowding remain poorly understood. In this work, we applied computational modeling to investigate the effects of macromolecular crowding on LLPS. To include biologically relevant LLPS dynamics, we extended the conventional Cahn–Hilliard model for phase separation by coupling it to experimentally derived macromolecular crowding dynamics and state-dependent reaction kinetics. Through extensive field-theoretic computer simulations, we show that the inclusion of macromolecular crowding results in late-stage coarsening and the stabilization of relatively smaller condensates. At a high crowding concentration, there is an accelerated growth and late-stage arrest of droplet formation, effectively resulting in anomalous labyrinthine morphologies akin to protein gelation observed in experiments. These results not only elucidate the crowder effects observed in experiments, but also highlight the importance of including state-dependent kinetics in LLPS models, and may help in designing further experiments to probe the intricate roles played by LLPS in self-organization dynamics of cells.

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Zhou, Huan-Xiang. "Effect of mixed macromolecular crowding agents on protein folding". Proteins: Structure, Function, and Bioinformatics 72, nr 4 (27.05.2008): 1109–13. http://dx.doi.org/10.1002/prot.22111.

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Biswas, Saikat, i Pramit K. Chowdhury. "Unusual domain movement in a multidomain protein in the presence of macromolecular crowders". Physical Chemistry Chemical Physics 17, nr 30 (2015): 19820–33. http://dx.doi.org/10.1039/c5cp02674a.

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Zhang, Chen, Fang Li, Si-Xi Wang, Zhao-Sheng Liu i Haji Akber Aisa. "Molecularly imprinted polymers prepared using a porogenic solvent of an ionic liquid and a macromolecular crowding agent and their application in purification of oleanic acid". Analytical Methods 7, nr 24 (2015): 10256–65. http://dx.doi.org/10.1039/c5ay01960e.

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A strategy to increase the affinity of molecularly imprinted polymers (MIPs) using a novel porogenic solvent was described based on the cooperative effect of ionic liquids and macromolecular crowding agents.

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Koch, Leon, Roland Pollak, Simon Ebbinghaus i Klaus Huber. "A Comparative Study on Cyanine Dyestuffs as Sensor Candidates for Macromolecular Crowding In Vitro and In Vivo". Biosensors 13, nr 7 (8.07.2023): 720. http://dx.doi.org/10.3390/bios13070720.

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Pseudo isocyanine chloride (PIC) has been identified in a preceding work as a sensor suited to probe macromolecular crowding both in test tubes with solutions of synthetic crowding agents and in HeLa cells as a representative of living systems. The sensing is based on a delicate response of the self-assembly pattern of PIC towards a variation in macromolecular crowding. Based on a suitable selection of criteria established in the present study, four additional cyanine dyestuffs (TDBC, S071, S2275, and PCYN) were scrutinized for their ability to act as such a sensor, and the results were compared with the corresponding performance of PIC. UV-VIS and fluorescence spectroscopy were applied to investigate the photo-physical properties of the four candidates and, if possible, light scattering was used to characterize the self-assembly of the dyestuffs in solution. Finally, HeLa cells were exposed to solutions of the most promising candidates in order to analyze their ability to infiltrate the cells and to self-assemble therein. None of the dyestuff candidates turned out to be as similarly promising in probing crowding effects in cells as PIC turned out to be. S0271 and S2275 are at least stable enough and meet the photophysical requirements necessary to act as sensors responding to changes in macromolecular crowding.

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Christopoulou, Natalia-Maria, Despina P. Kalogianni i Theodore K. Christopoulos. "Macromolecular crowding agents enhance the sensitivity of lateral flow immunoassays". Biosensors and Bioelectronics 218 (grudzień 2022): 114737. http://dx.doi.org/10.1016/j.bios.2022.114737.

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Rosin, Christopher, Paul Hendrik Schummel i Roland Winter. "Cosolvent and crowding effects on the polymerization kinetics of actin". Physical Chemistry Chemical Physics 17, nr 13 (2015): 8330–37. http://dx.doi.org/10.1039/c4cp04431b.

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Effects of cosolvents and macromolecular crowding agents on the G-to-F-transformation of actin are studied. Drastic and diverse changes in the lag phase and association rates of polymerizing actin are observed under different solvent conditions.

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Lin, Szu-Ning, Gijs J. L. Wuite i Remus T. Dame. "Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU". International Journal of Molecular Sciences 21, nr 24 (15.12.2020): 9553. http://dx.doi.org/10.3390/ijms21249553.

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HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

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Su, Wan-Chih, Douglas L. Gettel, Morgan Chabanon, Padmini Rangamani i Atul N. Parikh. "Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality". Journal of the American Chemical Society 140, nr 2 (5.01.2018): 691–99. http://dx.doi.org/10.1021/jacs.7b10192.

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Christiansen, Alexander, Qian Wang, Margaret S. Cheung i Pernilla Wittung-Stafshede. "Effects of macromolecular crowding agents on protein folding in vitro and in silico". Biophysical Reviews 5, nr 2 (19.02.2013): 137–45. http://dx.doi.org/10.1007/s12551-013-0108-0.

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Rowe, Ian, Andriy Anishkin, Kishore Kamaraju, Kenjiro Yoshimura i Sergei Sukharev. "The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding". Journal of General Physiology 143, nr 5 (28.04.2014): 543–57. http://dx.doi.org/10.1085/jgp.201311114.

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Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

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Mukherjee, Sanjib K., Saurabh Gautam, Saikat Biswas, Jayanta Kundu i Pramit K. Chowdhury. "Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study". Journal of Physical Chemistry B 119, nr 44 (23.10.2015): 14145–56. http://dx.doi.org/10.1021/acs.jpcb.5b09446.

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Dey, Pinki, i Arnab Bhattacherjee. "Disparity in anomalous diffusion of proteins searching for their target DNA sites in a crowded medium is controlled by the size, shape and mobility of macromolecular crowders". Soft Matter 15, nr 9 (2019): 1960–69. http://dx.doi.org/10.1039/c8sm01933a.

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Ferreira, L. A., V. N. Uversky i B. Y. Zaslavsky. "Role of solvent properties of water in crowding effects induced by macromolecular agents and osmolytes". Molecular BioSystems 13, nr 12 (2017): 2551–63. http://dx.doi.org/10.1039/c7mb00436b.

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Shahid, Sumra, Faizan Ahmad, Md Imtaiyaz Hassan i Asimul Islam. "Mixture of Macromolecular Crowding Agents Has a Non-additive Effect on the Stability of Proteins". Applied Biochemistry and Biotechnology 188, nr 4 (9.02.2019): 927–41. http://dx.doi.org/10.1007/s12010-019-02972-9.

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Paudel, Bishnu P., Erica Fiorini, Richard Börner, Roland K. O. Sigel i David S. Rueda. "Optimal molecular crowding accelerates group II intron folding and maximizes catalysis". Proceedings of the National Academy of Sciences 115, nr 47 (5.11.2018): 11917–22. http://dx.doi.org/10.1073/pnas.1806685115.

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Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.

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Rusinga, Farai I., i David D. Weis. "Automated Strong Cation-Exchange Cleanup To Remove Macromolecular Crowding Agents for Protein Hydrogen Exchange Mass Spectrometry". Analytical Chemistry 89, nr 2 (23.12.2016): 1275–82. http://dx.doi.org/10.1021/acs.analchem.6b04057.

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Li, Quan-Fu, Yan-Mei Zhan, Yong-Gang Zhong, Bo Zhang i Chang-Qing Ge. "Macromolecular Crowding Agents-Assisted Imprinted Polymers For Analysis Of Glycocholic Acid In Human Plasma And Urine". Biomedical Chromatography 30, nr 11 (20.05.2016): 1706–13. http://dx.doi.org/10.1002/bmc.3737.

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Shahid, Sumra, Ikramul Hasan, Faizan Ahmad, Md Imtaiyaz Hassan i Asimul Islam. "Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder". Biomolecules 9, nr 9 (12.09.2019): 477. http://dx.doi.org/10.3390/biom9090477.

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There are a large number of biomolecules that are accountable for the extremely crowded intracellular environment, which is totally different from the dilute solutions, i.e., the idealized conditions. Such crowded environment due to the presence of macromolecules of different sizes, shapes, and composition governs the level of crowding inside a cell. Thus, we investigated the effect of different sizes and shapes of crowders (ficoll 70, dextran 70, and dextran 40), which are polysaccharide in nature, on the thermodynamic stability, structure, and functional activity of two model proteins using UV-Vis spectroscopy and circular dichroism techniques. We observed that (a) the extent of stabilization of α-lactalbumin and lysozyme increases with the increasing concentration of the crowding agents due to the excluded volume effect and the small-sized and rod-shaped crowder, i.e., dextran 40 resulted in greater stabilization of both proteins than dextran 70 and ficoll 70; (b) structure of both the proteins remains unperturbed; and (c) enzymatic activity of lysozyme decreases with the increasing concentration of the crowder.

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Aumiller, William M., Bradley W. Davis, Emmanuel Hatzakis i Christine D. Keating. "Interactions of Macromolecular Crowding Agents and Cosolutes with Small-Molecule Substrates: Effect on Horseradish Peroxidase Activity with Two Different Substrates". Journal of Physical Chemistry B 118, nr 36 (26.08.2014): 10624–32. http://dx.doi.org/10.1021/jp506594f.

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Amzallag, Emmanuel, i Eran Hornstein. "Crosstalk between Biomolecular Condensates and Proteostasis". Cells 11, nr 15 (4.08.2022): 2415. http://dx.doi.org/10.3390/cells11152415.

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Proper homeostasis of the proteome, referred to as proteostasis, is maintained by chaperone-dependent refolding of misfolded proteins and by protein degradation via the ubiquitin-proteasome system and the autophagic machinery. This review will discuss a crosstalk between biomolecular condensates and proteostasis, whereby the crowding of proteostasis factors into macromolecular assemblies is often established by phase separation of membraneless biomolecular condensates. Specifically, ubiquitin and other posttranslational modifications come into play as agents of phase separation, essential for the formation of condensates and for ubiquitin-proteasome system activity. Furthermore, an intriguing connection associates malfunction of the same pathways to the accumulation of misfolded and ubiquitinated proteins in aberrant condensates, the formation of protein aggregates, and finally, to the pathogenesis of neurodegenerative diseases. The crosstalk between biomolecular condensates and proteostasis is an emerging theme in cellular and disease biology and further studies will focus on delineating specific molecular pathways involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases.

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Caterino, Tamara L., i Jeffrey J. Hayes. "Structure of the H1 C-terminal domain and function in chromatin condensationThis paper is one of a selection of papers published in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 89, nr 1 (luty 2011): 35–44. http://dx.doi.org/10.1139/o10-024.

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Linker histones are multifunctional proteins that are involved in a myriad of processes ranging from stabilizing the folding and condensation of chromatin to playing a direct role in regulating gene expression. However, how this class of enigmatic proteins binds in chromatin and accomplishes these functions remains unclear. Here we review data regarding the H1 structure and function in chromatin, with special emphasis on the C-terminal domain (CTD), which typically encompasses approximately half of the mass of the linker histone and includes a large excess of positively charged residues. Owing to its amino acid composition, the CTD was previously proposed to function in chromatin as an unstructured polycation. However, structural studies have shown that the CTD adopts detectable secondary structure when interacting with DNA and macromolecular crowding agents. We describe classic and recent experiments defining the function of this domain in chromatin folding and emerging data indicating that the function of this protein may be linked to intrinsic disorder.

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Chang, Jui-Yoa, Bao-Yun Lu i Por-Hsiung Lai. "Oxidative folding of hirudin in human serum". Biochemical Journal 394, nr 1 (27.01.2006): 249–57. http://dx.doi.org/10.1042/bj20051660.

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Human serum contains factors that promote oxidative folding of disulphide proteins. We demonstrate this here using hirudin as a model. Hirudin is a leech-derived thrombin-specific inhibitor containing 65 amino acids and three disulphide bonds. Oxidative folding of hirudin in human serum is shown to involve an initial phase of rapid disulphide formation (oxidation) to form the scrambled isomers as intermediates. This is followed by the stage of slow disulphide shuffling of scrambled isomers to attain the native hirudin. The kinetics of regenerating the native hirudin depend on the concentrations of both hirudin and human serum. Quantitative regeneration of native hirudin in undiluted human serum can be completed within 48 h, without any redox supplement. These results cannot be adequately explained by the existing oxidized thiol agents in human serum or the macromolecular crowding effect, and therefore indicate that human serum may contain yet to be identified potent oxidase(s) for assisting protein folding.

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Shahid, Sumra, Faizan Ahmad, Md Imtaiyaz Hassan i Asimul Islam. "Relationship between protein stability and functional activity in the presence of macromolecular crowding agents alone and in mixture: An insight into stability-activity trade-off". Archives of Biochemistry and Biophysics 584 (październik 2015): 42–50. http://dx.doi.org/10.1016/j.abb.2015.08.015.

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Vogel, Kristina, Thorsten Greinert, Monique Reichard, Christoph Held, Hauke Harms i Thomas Maskow. "Thermodynamics and Kinetics of Glycolytic Reactions. Part II: Influence of Cytosolic Conditions on Thermodynamic State Variables and Kinetic Parameters". International Journal of Molecular Sciences 21, nr 21 (25.10.2020): 7921. http://dx.doi.org/10.3390/ijms21217921.

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For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

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Werner, Tony E. R., Istvan Horvath i Pernilla Wittung-Stafshede. "Response to crowded conditions reveals compact nucleus for amyloid formation of folded protein". QRB Discovery 2 (2021). http://dx.doi.org/10.1017/qrd.2020.17.

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Abstract Although the consequences of the crowded cell environments may affect protein folding, function and misfolding reactions, these processes are often studied in dilute solutions in vitro. We here used biophysical experiments to investigate the amyloid fibril formation process of the fish protein apo-β-parvalbumin in solvent conditions that mimic steric and solvation aspects of the in vivo milieu. Apo-β-parvalbumin is a folded protein that readily adopts an amyloid state via a nucleation–elongation mechanism. Aggregation experiments in the presence of macromolecular crowding agents (probing excluded volume, entropic effects) as well as small molecule osmolytes (probing solvation, enthalpic effects) revealed that both types of agents accelerate overall amyloid formation, but the elongation step was faster with macromolecular crowding agents but slower in the presence of osmolytes. The observations can be explained by the steric effects of excluded volume favoring assembled states and that amyloid nucleation does not involve monomer unfolding. In contrast, the solvation effects due to osmolyte presence promote nucleation but not elongation. Therefore, the amyloid-competent nuclei must be compact with less osmolytes excluded from the surface than either the folded monomers or amyloid fibers. We conclude that, in contrast to other amyloidogenic folded proteins, amyloid formation of apo-β-parvalbumin is accelerated by crowded cell-like conditions due to a nucleation process that does not involve large-scale protein unfolding.

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Okamoto, Debora N., Lilian C. G. Oliveira, Marcia Y. Kondo, Maria H. S. Cezari, Zoltán Szeltner, Tünde Juhász, Maria A. Juliano, László Polgár, Luiz Juliano i Iuri E. Gouvea. "Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition". Biological Chemistry 391, nr 12 (1.12.2010). http://dx.doi.org/10.1515/bc.2010.145.

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Abstract The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na2SO4, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K d) indicated that activation was a result of a more active dimer, without significant changes in K d values. The activation was found to be independent of substrate length and was derived from both k cat increase and K m decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.

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Lecinski, Sarah, Jack W. Shepherd, Kate Bunting, Lara Dresser, Steven D. Quinn, Chris MacDonald i Mark C. Leake. "Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo". Interface Focus 12, nr 6 (14.10.2022). http://dx.doi.org/10.1098/rsfs.2022.0042.

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In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

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Louisthelmy, Rebecca, Brycen M. Burke i R. Chase Cornelison. "Brain cancer cell-derived matrices and effects on astrocyte migration". Cells Tissues Organs, 15.02.2022. http://dx.doi.org/10.1159/000522609.

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Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of macromolecular crowding (MMC) alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously-reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

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Hagedoorn, Peter-Leon. "Isothermal Titration Calorimetry in Biocatalysis". Frontiers in Catalysis 2 (10.05.2022). http://dx.doi.org/10.3389/fctls.2022.906668.

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Isothermal titration calorimetry (ITC) is a popular chemical analysis technique that can be used to measure macromolecular interactions and chemical and physical processes. ITC involves the measurement of heat flow to and from a measurement cell after each injection during a titration experiment. ITC has been useful to measure the thermodynamics of macromolecular interactions such as protein-ligand or protein-protein binding affinity and also chemical processes such as enzyme catalyzed reactions. The use of ITC in biocatalysis has a number of advantages as ITC enables the measurement of enzyme kinetic parameters in a direct manner and, in principle, can be used for most enzymes and substrates. ITC approaches have been developed to measure reversible and irreversible enzyme inhibition, the effects of molecular crowding on enzyme activity, the activity of immobilized enzymes and the conversion of complex polymeric substrates. A disadvantage is that in order to obtain accurate kinetic parameters special care has to be taken in proper experimental design and data interpretation, which unfortunately is not always the case in reported studies. Furthermore, special caution is necessary when ITC experiments are performed that include solvents, reducing agents and may have side reactions. An important bottleneck in the use of calorimetry to measure enzyme activity is the relatively low throughput, which may be solved in the future by sensitive chip based microfluidic enzyme calorimetric devices.

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Siraj, Seerat, Daraksha Yameen, Anas Shamsi, Faizya Khan, Asimul Islam i Mohammad Mahfuzul Haque. "Interaction of Thioflavin T (ThT) and 8-anilino-1-naphthalene sulfonic acid (ANS) with macromolecular crowding agents and their monomers: Biophysical analysis using in vitro and computational approaches". Journal of Molecular Liquids, styczeń 2023, 121270. http://dx.doi.org/10.1016/j.molliq.2023.121270.

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Bonucci, Alessio, Martina Palomino-Schätzlein, Paula Malo de Molina, Arantxa Arbe, Roberta Pierattelli, Bruno Rizzuti, Juan L. Iovanna i José L. Neira. "Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1". Frontiers in Molecular Biosciences 8 (5.07.2021). http://dx.doi.org/10.3389/fmolb.2021.684622.

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The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

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Köhn, Birgit, i Michael Kovermann. "All atom insights into the impact of crowded environments on protein stability by NMR spectroscopy". Nature Communications 11, nr 1 (13.11.2020). http://dx.doi.org/10.1038/s41467-020-19616-w.

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AbstractThe high density of macromolecules affecting proteins due to volume exclusion has been discussed in theory but numerous in vivo experiments cannot be sufficiently understood taking only pure entropic stabilization into account. Here, we show that the thermodynamic stability of a beta barrel protein increases equally at all atomic levels comparing crowded environments with dilute conditions by applying multidimensional high-resolution NMR spectroscopy in a systematic manner. Different crowding agents evoke a pure stabilization cooperatively and do not disturb the surface or integrity of the protein fold. The here developed methodology provides a solid base that can be easily expanded to incorporate e.g. binding partners to recognize functional consequences of crowded conditions. Our results are relevant to research projects targeting soluble proteins in vivo as it can be anticipated that their thermodynamic stability increase comparably and has consequently to be taken into account to coherently understand intracellular processes.

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