Arc Repressor will not Denature Under Pressure in the Absence of Water

Oliveira, Andréa C.; Gaspar, Luciane P.; Poian, Andrea T. Da; Silva, Jerson L.

doi:10.1006/jmbi.1994.1433

Cited by 95 publications

(70 citation statements)

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“…A faster rotation in the nonpolar core of the empty capsids suggests a more flexible conformation in the absence of the protein-RNA interactions, which supports our previous data on the increasing facility to denature this form to a molten-globule structure. Indeed, the Arc repressor dimer, which is also denatured by hydrostatic pressure to a molten globule conformation (34), is gradually stabilized by increasing glycerol concentration in the medium, suggesting that a loss of structural flexibility promoted by decreasing water concentration makes it more difficult for the protein to denature to a molten globule conformation (40). The interactions between proteins and nucleic acid in viral capsids probably lead to a similar loss in structural flexibility, resulting in a great stabilization, which is crucial to protection against extracellular environments.…”

Section: Discussionmentioning

confidence: 99%

“…High pressure provides a remarkable way to evaluate the relation between dynamics and stability (17,18), especially because of the direct relation between protein flexibility (due to volume fluctuations) and its isothermal compressibility (39). Pressure is well known to perturb the native state of proteins by causing the infiltration of molecules of water (17,40,41). Fig.…”

Section: Fluorescence Properties Of Cpmvmentioning

confidence: 99%

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Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Poian

Johnson

Silva

2002

Journal of Biological Chemistry

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The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and timeresolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, timeresolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.Virus assembly is a puzzle when viewed from the perspectives of thermodynamics and kinetics. Virus assembly touches questions related to protein folding, protein-protein interactions, and protein-nucleic acid interactions. As previously shown for several viruses, these questions are quite linked in many bacteria, plant, and animal viruses (1-5). Although much has been learned about the structure of many macromolecular assemblies, in most cases the direct correlation with function is not possible because of the lack of information about the dynamics of the system (4). In solution, icosahedral viruses can be viewed as molecular "quasicrystals," and they are good models for understanding the linkage among protein folding, protein-nucleic acid interactions, and macromolecular assembly.Comoviridae is a family of icosahedral plant viruses characterized by a divided genome. This genome consists in two single-stranded, positive-sense RNA molecules, which are encapsidated into distinct particles. The capsids contain equimolar amounts of a large (L) and a small (S) proteins. 1 The two RNA molecules are referred to as RNA 1 and RNA 2, with ϳ6.0 and 3.5 kb, respectively (6). Isolation of comovirus from infected plants results in a mixture of the two ribonucleoprotein particles, as well as empty shells. Thus, three different particles can be separated by gradient ultracentrifugation from preparations of these viruses: the top (no RNA), the middle (containing RNA 2), and the bottom (c...

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Section: Discussionmentioning

confidence: 99%

Section: Fluorescence Properties Of Cpmvmentioning

confidence: 99%

Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Poian

Johnson

Silva

2002

Journal of Biological Chemistry

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“…ducing the penetration of molecules of water into the interior of the protein structure (52,53), which facilitates the population of partially folded segments of the protein. This "shaking" effect on the protein structure makes the N state of the envelope proteins jump into the FG state.…”

Section: Fig 5 Pressure-induced Fusogenic State Of Sindbis Virusmentioning

confidence: 99%

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Gaspar¹,

Silva²,

Gomes³

et al. 2002

Journal of Biological Chemistry

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Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.Enveloped viruses utilize regulated membrane fusion to introduce their genomes in the cytoplasm of the host cell. The fusion is mediated by surface envelope proteins of the virus in response to a trigger (1, 2). Once triggered, the fusion process leads to a conformational change that promotes the interaction of a specific sequence (fusion peptide) with the target membrane and initiates membrane fusion. Membrane fusion is crucial in other biological functions such as myotube formation, fertilization, and trafficking of endocytic and exocytic vesicles within eukaryotic cells (1, 3). Many enveloped animal viruses have been studied as models for understanding the mechanism of membrane fusion. While the fusion proteins of many viruses reveal significant similarity in their putative fusogenic conformation, such as those of influenza virus (hemagglutinin HA2), 1 human immunodeficiency virus (gp41 protein), Moloney murine leukemia virus (TM protein), and Ebola virus (GP2 protein) (4), the events of membrane fusion for other virus families (e.g. Flaviviridae and Togaviridae) are beginning to be understood. Alphavirus and the flavivirus fusion proteins appear to have evolved from a common ancestor (5) and possess a similar new class of membrane fusion proteins that do not form coiledcoils (6 -8).Sindbis and influenza are enveloped viruses that first enter a cell by endocytosis and then fuse with the cellular membrane in response to acidic conditions. Sindbis virus is the prototype of the Alphavirus genus, Togaviridae family. The Alphavirus spike is...

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“…Several studies have highlighted the crucial role of water in the pressure-induced denaturation of biological systems. Oliveira et al (28) reported that protein denaturation decreased linearly with a decrease in water concentration. Similarly, Kinsho et al (13) observed that the removal of water by the addition of polyols or small cationic ions had an efficient protective effect against enzyme inactivation at high pressures and subzero temperatures.…”

Section: Discussionmentioning

confidence: 99%

Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation ofEscherichia coli

Moussa

Perrier-Cornet

Gervais

2006

Appl Environ Microbiol

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The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (؊20°C without freezing and 25°C) and three water activity levels (a w ) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at ؊20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in the range of 100 to 300 MPa with an a w of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a w was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.Food processing under high hydrostatic pressure is an emerging technology that has stimulated considerable interest within the food industry over the past 15 years. There are currently some interesting commercial opportunities and research challenges in the high-pressure processing of foods (42). This processing technique allows the manufacture of innovative food products while preserving the texture, color, raw flavoring agents, and nutritional value of the food, which are all aspects valued by consumers (22,36). Furthermore, high-pressure treatments cause the denaturation of several enzymes that are responsible for quality deterioration, as well as the inactivation of pathogenic and spoilage microorganisms (15). However, to achieve high or complete microbial inactivation, high pressures and/or long treatment times are required, so that the cost of the process seriously limits its industrial applications. For this reason, it would be beneficial to optimize high-hydrostatic-pressure processes.Numerous studies have demonstrated the temperature dependence of the antimicrobial effects of high pressure (19,25,39). Moreover, the efficiency of high-pressure treatments is controlled by other process parameters such as the applied pressure and the kinetics of pressurization (31), as well as by the physicochemical properties of the medium being treated, such as pH (1, 18) and water activity (10,26,43). Precise control of these parameters is necessary to ...

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Arc Repressor will not Denature Under Pressure in the Absence of Water

Cited by 95 publications

References 0 publications

Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation ofEscherichia coli

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