ABSTRACT
Zearalenone and its cis-isomer, cis-zearalenone, are nonsteroidal mycotoxins that elicit an estrogenic response upon binding to the estrogen receptor. This study compares the estrogenicity of eleven congeners including novel metabolites as 15-OH-zearalenone, zearalenone-14-sulfate, α-cis-zearalenol and ß-cis-zearalenol using the E-Screen assay. Overall, a change in the configuration from trans to cis retains significant estrogenic activity. In contrast, alterations of the aromatic moiety including hydroxylation and sulfation showed a markedly decreased estrogenicity when compared to zearalenone.
Subject(s)
Estrogens/metabolism , Zearalenone/metabolism , Cell Line, Tumor , HumansABSTRACT
Specific helix-helix interactions underpin the correct assembly of multipass membrane proteins. Here, we show that a designed buried salt bridge mediates heterodimer formation of model transmembrane helical peptides in a pH-dependent manner. The model peptides bear side chains functionalized with either a carboxylic acid or a primary amine within a hydrophobic segment. The association behavior was monitored by Förster resonance energy transfer, revealing that heterodimer formation is maximized at a pH close to neutrality (pH 6.5), at which each peptide is found in a charged state. In contrast, heterodimerization is disfavored at low and high values of pH, because either the carboxylic acid or the primary amine is present in its neutral state, thus preventing the formation of a salt bridge. These findings provide a blueprint for the design and modulation of protein-protein interactions in membrane proteins.
Subject(s)
Cell Membrane/chemistry , Peptides/chemistry , Protein Multimerization , Salts/chemistry , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Fluorescence Resonance Energy Transfer , Hydrogen-Ion Concentration , Molecular Sequence Data , Peptides/chemical synthesis , Protein Structure, SecondaryABSTRACT
Sliding clamps are ring-shaped oligomeric proteins that are essential for processive deoxyribonucleic acid replication. Although crystallographic structures of several clamps have been determined, much less is known about clamp structure and dynamics in solution. Here, we characterized the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli ß and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) clamps using single-molecule approaches. We show that E. coli ß is stable in solution as a closed ring at concentrations three orders of magnitude lower than PCNA. The trimeric structure of PCNA results in slow subunit association rates and is largely responsible for the lower solution stability. Despite this large difference, the intrinsic lifetimes of the rings differ by only one order of magnitude. Our results show that the longer lifetime of the E. coli ß dimer is due to more prominent electrostatic interactions that stabilize the subunit interfaces.
Subject(s)
DNA Polymerase III/metabolism , Proliferating Cell Nuclear Antigen/metabolism , DNA Polymerase III/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Protein Multimerization , Protein Subunits , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Spectrometry, FluorescenceABSTRACT
Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).
Subject(s)
Darkness , Luminescent Proteins/chemistry , Luminescent Proteins/radiation effects , Photolysis , Calorimetry, Differential Scanning , Kinetics , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/radiation effects , Mutation/genetics , Numerical Analysis, Computer-Assisted , Photobleaching/radiation effects , Protein Conformation/radiation effects , Spectrometry, Fluorescence , Red Fluorescent ProteinABSTRACT
We characterized the effect of the first basepair on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of double-stranded DNA using gaussian-mixture adaptive umbrella sampling simulations. In the simulations, the sampling of all five dihedral angles along the linker was enhanced, so that both stacked and unstacked states were sampled. The affinity of Cy3 for a T·A basepair (with the dye attached to T) was found to be significantly less than for the other basepairs. This was verified experimentally by measuring the activation energies for cis-trans isomerization of the dye. The simulation and experimental results indicate the existence of partially unstacked conformations amenable to photoisomerization. The simulations also showed that stacking of Cy3 straightens the DNA while stabilizing the first basepair. Our findings indicate that fluorescence is modulated by Cy3-DNA interactions in a sequence-dependent manner.
