Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~ (12+2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered

Synthesis and Photochemistry of a New Class of Photocleavable ProteinCross-linking Reagents

[eng] A new series of photocleavable protein cross-linking reagents based on bis(maleimide) derivatives of diaryl disulfides have been synthesised. They have been functionalised with cysteineand transient absorption spectra for the photolysis reaction have been recorded by using the pump-probe techniquewith a time resolution of 100 femtoseconds. Photolysis of the disulfide bond yields the corresponding thiyl radicalsin less than a picosecond. There is a significant amount of geminate recombination,but some of the radicalsescape the solvent cage and the quantumyield for photocleavage is 30% in water

Measurement of energy landscape roughness of folded and unfolded proteins

[eng] The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow themotion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting froma nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in closecontact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us toquantify the roughness of the energy landscape (4–5 kBT). Surprisingly,even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 kBT) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme

The Appearance of Stochastic Control in Fish Communities on Coral Reefs: a Hierarchical Approach to System Organization

Species membership and population densities of fish communities on coral reefs are highly variable both spatially and temporally across many scales. Several models of stochastic control (i.e., organizing processes that produce these highly variable patterns) have been proposed. These models of stochastic control include: a lottery for available habitat sites, limited recruitment, and opportunistic predation. Although these models of stochastic control differ from each other in their organizing processes, they are similar in that they share two implicit assumptions. First, there is only one level or scale of system organization; second, there is only one significant organizing process. Recent developments in community ecology, however, suggest that the organization of fish communities on coral reefs is complex (i.e., involves multiple levels of system organization). The heuristic value of a multi-level system of organization is that patterns that appear under stochastic control can, at least theoretically, be broken down or decomposed into a series of simpler, non-stochastic patterns. If fish communities on coral reefs are indeed complex as in the above sense, then we need to re-evaluate the supposed single-level stochastic control in these systems. The purpose of this thesis, therefore, is to test the null hypothesis that fish communities on coral reefs are dependent on single-level stochastic processes as has been suggested. The alternative hypothesis is the appearance of stochastic control is a blend of patterns due to multiple levels of system organization. The method I use to test this is conceptually straightforward. The first step is to divide the species membership into several groups. In the second step, for each species group I determine the correlation between species distributions and, for example, some attribute of the habitat template. If the organization of fish communities on coral reefs is dependent on stochastic processes acting within a single level, then independent of how the species are divided into groups, the correlation for all species groups should be similar. I found that: - when the species are divided into groups based on similarities in the number of patch reefs a species is found, then significant differences occur among groups in correlation between species distributions and habitat attributes, - specifically, for each habitat attribute tested there exists a trend of increasing correlation (or decreasing correlation -- depending on the habitat attribute) from species found on most patches to species found on few patches. This suggests that the previous models of single-level stochastic control (e.g., habitat lottery, limited recruitment, and predation) are incomplete because they do not consider consequences due to differences in species' distributions and densities. Instead these results suggest that the structure and organization of fish on coral reefs incorporate a mixture of stochastic and non-stochastic processes that occur (a) simultaneously within the community, and (b) differentially between the species. This implies that fish communities on coral reefs are complex concerning their basic structure, and that unless a community level pattern is decomposed into its component elements, the conclusions made about the nature of these communities can be misleading.Doctor of Philosophy (PhD