A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alphabeta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alphabeta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alphabeta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alphabeta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting 'competitive' antagonist) binds to this residue.     

Progression into and out of mitosis

Progression through mitosis is controlled by cyclin-dependent kinases, which drive cells into metaphase, and by the anaphase-promoting complex/cyclosome, a ubiquitin ligase that triggers sister chromatid separation and exit from mitosis. Recent work has shown how the mutual regulation between cyclin-dependent kinases and the anaphase-promoting complex/cyclosome ensures that cell-cycle events occur in the right order. The analysis of complexes required for sister chromatid cohesion and chromosome condensation has revealed how cyclin-dependent kinases and the anaphase-promoting complex/cyclosome control the behaviour of chromosomes.     

Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H<Subscript>2</Subscript>O<Subscript>2</Subscript> exposure

Summary. The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.     

Positioning the genome within the nucleus

Higher eukaryotic genomes contain both housekeeping genes and genes of which the expression is restricted to a defined time and space. It is well established that a correlation exists between structural organization of the genome and gene expression control. The functional mechanisms underlying this correlation are still poorly understood. Here I describe several observations that are the basis of present concepts of genome organization and nuclear architecture related to functionality. Regarding the relationship between positioning and disturbed cell functionality, I describe observations showing that the proximity of selected gene loci is statistically correlated with their propensity for oncogenic translocations as well as observations of patterns occurring in neurodegenerative disorders where unstable repeats are translated into an expanded polyglutamine tract. Such observations underscore the importance to understand how genetic perturbations lead to the global reorganization of nuclear architecture, chromatin structure and widespread changes in gene expression.     

Structural conservation in the major facilitator superfamily as revealed by comparative modeling

The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published. Despite the low sequence similarity of the two proteins involved, lactose permease and glycerol-3-phosphate transporter, the reported structures are highly similar. This leads to the hypothesis that all members of the MFS share a similar structure, regardless of their low sequence identity. To test this hypothesis, we generated models of two other members of the MFS, the Tn10-encoded metal-tetracycline/H(+) antiporter (TetAB) and the rat vesicular monoamine transporter (rVMAT2). The models are based on the two MFS structures and on experimental data. The models for both proteins are in good agreement with the data available and support the notion of a shared fold for all MFS proteins.     

Development of a fluorescent probe for the study of nucleosome assembly and dynamics

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.