Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

Foguel, Débora; Suarez, Marisa C.; Barbosa, Catão T.F.; Rodrigues, J J; Sorenson, Martha M.; Smillie, Lawrence B.; Silva, Jerson L.

doi:10.1073/pnas.93.20.10642

Cited by 39 publications

(51 citation statements)

References 34 publications

(32 reference statements)

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“…In this way, one might expect ⌬N W to contribute more importantly to dln K obs /dln a W than does ⌬N glycerol . A similar conclusion was reached for other systems in which dln K obs /dln a W was obtained with glycerol and other osmolytes that differ structurally and chemically (37,38,48,54,55).…”

Section: Discussionsupporting

confidence: 80%

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Lima

Silva

2004

Journal of Biological Chemistry

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Protein-nucleic acid interactions are responsible for the regulation of key biological events such as genomic transcription and recombination and viral replication. However, the recognition mechanisms involved in these processes are not completely understood. Here, we investigate the dominant forces involved in protein-protein and protein-DNA interactions for the 80-amino-acid C-terminal domain of the E2 protein (E2c) from human papillomavirus (HPV-16). The E2c protein is a homodimer that specifically binds to double-stranded DNA containing the consensus sequence ACCG-N 4 -CGGT, where N is any nucleotide. DNA binding affinity is reduced by lowering water chemical potential, accompanied by an increase in cooperativity. Wyman linkage relations between affinity and water chemical potential indicate that 11 additional water molecules are bound in the formation of the complex between E2c and DNA. Salt dissociation isotherms showed that 10 counterions are released upon association, even at low water activity, indicating that this latter variable does not change the electrostatic component of the interaction. Further analysis demonstrates a strong dependence of cooperativity of binding on the protein concentration. Altogether, these results reveal a novel binding pathway in which the consolidated complex may achieve its final form via a monomer-DNA intermediate, which favors the binding of a second monomer. This molecular mechanism reveals the contributions of multiple conformers in a tight virus genome modulation that seems to be important in the cell infection scenario. Human papillomavirus (HPV)1 is a small nonenveloped double-stranded DNA virus that causes epithelial lesions, which often develop into malignancies (1). The viruses HPV 16, -18, and -31 are denoted high risk HPV since they are directly related to cervical cancer. The regulation of gene expression in virus-infected cells is mediated by the factor E2, which controls the transcription of the HPV gene, including those related to malignant transformation. E2 can act either as an activator or as a repressor, depending on the alternate splicing of the product. Infected cells can be found containing (i) the C-terminal domain (E2c), responsible for DNA binding (2) and dimerization (3); (ii) the E2-E8 splicing product; and (iii) the full E2 protein (E2-full), consisting of the E2c, the N-terminal transactivation domain (E2TA), and an unstructured region with little known function. These three components recognize and bind specifically the palindromic sequence ACCGN 4 CGGT (2), where N is any base, although differences in affinity can arise depending on the N bases (4 -7). Part of the E2 structure has been solved: both for E2c, the DNA-binding domain (7-10), and for the transactivation domain (11,12).Much has appeared in the literature about the behavior of the E2c protein from the high risk HPV-16, in terms of homoassociation (13-16) and DNA binding and recognition, through equilibrium (6, 9, 14, 16 -20) and kinetic assays (4, 21). Although we have previously p...

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Section: Discussionsupporting

confidence: 80%

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Lima

Silva

2004

Journal of Biological Chemistry

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“…Fluorescent probes engineered into TnC through Phe to Trp mutation in site I have been used previously to study the Ca 2ϩ binding dynamics of this molecule (8,19,21,27,29,32,38). We have demonstrated the effectiveness of Trp at residue 27 in reporting Ca 2ϩ binding to site II in McTnC and ScTnC without significantly affecting the ␣-helical content of the protein, which reflects the general structure of the molecule, using far UV circular dichroism spectra (27).…”

Section: Discussionmentioning

confidence: 98%

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca²⁺ affinity of site II

Gillis

Tibbits

2003

American Journal of Physiology-Cell Physiology

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Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca2+ than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca2+ sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca2+ affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca2+ affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca2+ binding to site II (F27W). The removal of the COOH terminus (amino acids 90–161) from ScTnC and McTnC maintained the difference in Ca2+ affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln29 and Asp30 in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca2+ affinity to that of McNTnC. These results demonstrate that Gln29 and Asp30 in ScTnC are required for the high Ca2+ affinity of site II.

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“…Pressure shifts any association/dissociation (or folding/ unfolding) reaction toward the smaller volume whether in monomeric proteins (17,44,45), in oligomeric proteins (16,18,37), in protein-DNA complexes (46 -49), or in the conversion of proteins into the amyloidogenic or aggregated state (50,51). In all these cases, pressure appears to produce its effects by in -FIG.…”

Section: Fig 3 Pressure Effects On Sindbis and Influenza Virusesmentioning

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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Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

Cited by 39 publications

References 34 publications

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca²⁺ affinity of site II

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

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Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

Cited by 39 publications

References 34 publications

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

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Sequence mutations in teleost cardiac troponin C that are permissive of high Ca²⁺ affinity of site II