Broad spectrum light and night-time mental performance : effects of intensity and duration : a thesis completed in partial fulfilment of the requirements for the degree of Master of Arts

The present study examined the effects of light intensity and duration on mental performance at night. A number of investigations have found light levels as low as 500 lux can have a significant impact on cognition, but there have been few, if any, systematic experiments that have investigated the potential trade-off between the intensity of the light and its duration. Light levels of 100 (normal room lighting), 300, 600 and 1,000 lux were paired with one of two different light exposure times: 15 and 60 minutes. Sixteen volunteers completed tests of critical thinking, simple maths, letter cancellation, recall, and recognition between 2300 and 0100 hours once a week for four consecutive weeks. Body temperature and subjective sleepiness levels were also recorded. The results showed that, in general, light intensities, irrespective of duration, of 300 and 600 lux had a positive effect on critical thinking and recognition memory. In contrast to some previous findings, there was little or no effect on sleepiness levels, core body temperature, recall, letter cancellation or the simple maths task. Surprisingly, the 1,000 lux light level had no effect on any of the tasks. It was concluded that changes in the intensity of broad-spectrum light can affect night-time cognitive performance, but that the intensity of the light cannot be traded for duration. However, further investigation of the manner in which light intensity is varied, either by distance from the light or by varying the brightness of the light source, is required before firm conclusions can be drawn

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNA-binding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.United States. Public Health Service (GM049224

Proteolysis in the Escherichia coli heat shock response: a player at many levels

Proteolysis is a fundamental process used by all forms of life to maintain homeostasis, as well as to remodel the proteome following environmental changes. Here, we explore recent advances in understanding the role of proteolysis during the heat shock response of Escherichia coli. Proteolysis both regulates and contributes directly to and the heat shock response at multiple different levels, from adjusting the levels of the master heat shock response regulator (σ[superscript 32]), to eliminating damaged cellular proteins, to altering the activity of chaperones that refold heat-denatured proteins. Recent results illustrate the complexity of the heat shock response and the pervasive role that proteolysis plays in both the cellular response to heat shock and the subsequent limiting of the response, as cells return to a more ‘normal’ physiological state

Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.National Institutes of Health (U.S.) (Grant AI-16892

Assembly of phage Mu transpososomes: Cooperative transitions assisted by protein and DNA scaffolds

AbstractTransposition of phage Mu takes place within higher order protein-DNA complexes called transpososomes. These complexes contain the two Mu genome ends synapsed by a tetramer of Mu transposase (MuA). Transpososome assembly is tightly controlled by multiple protein and DNA sequence cofactors. We find that assembly can occur through two distinct pathways. One previously described pathway depends on an enhancer-like sequence element, the internal activation sequence (IAS). The second pathway depends on a MuB protein-target DNA complex. For both pathways, all four MuA monomers in the tetramer need to interact with an assembly-assisting element, either the IAS or MuB. However, once assembled, not all MuA monomers within the transpososome need to interact with MuB to capture MuB-bound target DNA. The multiple layers of control likely are used in vivo to ensure efficient rounds of DNA replication when needed, while minimizing unwanted transposition products

Stepwise Unfolding of a β Barrel Protein by the AAA+ ClpXP Protease

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.National Institutes of Health (U.S.) (Grant AI-15706

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.National Institutes of Health (U.S.) (Grant AI-16982)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Experiencing education in the new Christian schools in the United Kingdom : listening to the male graduates

The new independent Christian schools developed by parents and evangelical churches in the United Kingdom since the late 1960s remain controversial among both Christian and secular educators. In response to this controversy, the present study traced 106 men who had graduated from these schools between 1985 and 2003 and analysed their evaluation of the education they had received in these schools within four main themes: the quality of the education, the context of Christian and moral nurture, the quality of relationships (among the pupils, with the teachers and with the wider world) and preparation received for life after leaving school. Although there were some issues of criticism, the balance of opinion among the former pupils within all four areas was generally supportive of the new independent Christian schools, which were generally perceived as having prepared them well for life

Steric clashes with bound OMP peptides activate the DegS stress-response protease

Escherichia coli senses envelope stress using a signaling cascade initiated when DegS cleaves a transmembrane inhibitor of a transcriptional activator for response genes. Each subunit of the DegS trimer contains a protease domain and a PDZ domain. During stress, unassembled outer-membrane proteins (OMPs) accumulate in the periplasm and their C-terminal peptides activate DegS by binding to its PDZ domains. In the absence of stress, autoinhibitory interactions, mediated by the L3 loop, stabilize inactive DegS, but it is not known how this autoinhibition is reversed during activation. Here, we show that OMP peptides initiate a steric clash between the PDZ domain and the L3 loop that results in a structural rearrangement of the loop and breaking of autoinhibitory interactions. Many different L3-loop sequences are compatible with activation but those that relieve the steric clash reduce OMP activation dramatically. Our results provide a compelling molecular mechanism for allosteric activation of DegS by OMP-peptide binding.National Institutes of Health (U.S.) (Grant AI-16892