Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ2, χ3, χ2′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ2 − 60°, χ390, χ2′ − 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.

Estimating relative disulfide energies : an accurate ab initio potential energy surface

Disulfide torsional energy, a good predictor of disulfide redox potential in proteins, may be estimated by interpolation on a potential energy surface (PES) describing the twisting of diethyl disulfide through its three central dihedral angles. Here we update PES calculations at the M05-2X level of theory with the 6-31G(d) basis set. Although the surface shows no qualitative differences from an earlier MP2(full) PES, energy differences greater than 1 kJ mol^–1 were seen for conformations with χ₂ between –60° and 30°, or with χ₃ below 60° or above 130°. This is particularly significant for highly strained disulfides that are likely to be spontaneously reduced by mechanical means. In benchmarking against the high-level G3X method, M05-2X showed significantly reduced root mean squared deviation compared with MP2(full) (1.0 versus 2.0 kJ mol^–1 respectively). Results are incorporated into a web application that calculates relative torsional energies from disulfide dihedral angles (http://www.sbinf.org/applications/pes.html).

The 'caffeine-sweetness' effect; potential reduction of energy in caffeinated sugar-sweetened soft drinks

Background – Excessive consumption of sugar sweetened beverages (SSB) is a contributing factor in the occurrence of overweight and obesity. The high energy intake, low satiation, high glycemic index, and intense marketing are all thought to contribute to their over consumption. In addition, the role of the mildly-addictive chemical caffeine in SSB has been questioned (Griffiths and Vernotica, 2000, Keast and Riddell, 2007). We have previously shown that low concentrations of caffeine may decrease sweetness of sugars and thereby result in excess energy in SSB formulations (Ebbeling et al., 2006).
Objective – Without noticeably affecting flavour, to determine potential energy reduction when decreasing sucrose concentration from caffeinated and de-caffeinated SSB.
Design – Human psychophysical taste evaluations in water, sucrose and model SSB. Triangle forced-choice ascending method of limits was used to determine caffeine taste threshold in water and sucrose (n= 62). Directional paired comparison tests to determine 1/ the influence of caffeine on sweetness of sucrose (n= 23), and 2/ the nonperceivable difference when decreasing the sucrose and caffeine concentrations in a model SSB (n= 30).
Outcomes – Caffeine, at sub-threshold concentrations in common SSB (0.67mM) can be perceived in sucrose solutions because it significantly inhibits sweetness (p<0.001), the ‘caffeine sweetness effect’. Presumably coremoval of caffeine and sucrose could be achieved without affecting the sweetness of the SSB. Removing caffeine from the model SSB allowed an energy reduction of 137.4 KJ per 500 ml serving (12.6% sucrose reduction) without noticeably affecting flavour for 80% of the population. The energy reduction possible without co-removal of caffeine was a more modest 32 KJ per 500 ml serving (3.5% sucrose reduction).
Conclusion – Sub-threshold concentrations of caffeine suppress sweetness resulting in higher concentrations of sugars in SSB. Excessive consumption of SSB is linked to the obesity epidemic, and we suggest the removal of caffeine and subsequent removal of 137.4 KJ energy will have long term public health benefits.

Laboratory model studies of flushing of trapped salt water from a blocked tidal estuary

Results are presented from a series of laboratory model studies of the flushing of saline water from a partially- or fully-closed estuary. Experiments have been carried out to determine quantitatively the response of the trapped saline volume to fresh water flushing discharges Q for different values of the estuary bed slope α and the density difference (∆ρ)_o between the saline and fresh water. The trapped saline water forms a wedge within the estuary and for maintained steady discharges, flow visualisation and density profile data confirm that its response to the imposition of the freshwater purging flow occurs in two stages, namely (i) an initial phase characterised by intense shear-induced mixing at the nose of the wedge and (ii) a relatively quiescent second phase where the mixing is significantly reduced and the wedge is forced relatively slowly down and along the bed slope. Scalings based upon simple energy balance considerations are shown to be successful in (i) describing the time-dependent wedge behaviour and (ii) quantifying the proportion of input kinetic energy converted into increasing the potential energy of the wedge/river system. Measurements show that the asymptotic value of the energy conversion factor increases with increasing value of the river Froude number Fr_o at small values of Fr_o, thereafter reaching a maximum value and a gradual decrease at the highest values of Fr_o. Dimensional analysis considerations indicate that the normalised, time-dependent wedge position (x_w)³(g')o/q² can be represented empirically by a power-law relationship of the form (x_w)[(g')_o/q²]^1/3 =C [(t)[(g')_o²/q]^1/3]"where the proportionality coefficient C is a function of both Fr_o and the slope angle α and the exponent n has a value of 0.24. Successful attempts are made to relate the model data to existing field observations from a microtidal estuary.

Experiments with multiple, intermittent periodic flushing flows confirm the importance of the starting phase of each flushing event for the time dependent behaviour of the saline wedge after reaching equilibrium in the intervals between such events. For the parameter ranges investigated and for otherwise-identical external conditions, no significant differences are found in the position of the wedge between cases of sequential multiple flushing flows and steady single discharges of the same total duration.

Predicting molecular structures: an application of the cutting angle method

The ability to predict molecular geometries has important applications in chemistry. Specific examples include the areas of protein space structure elucidation, the investigation of host–guest interactions, the understanding of properties of superconductors and of zeolites. This prediction of molecular geometries often depends on finding the global minimum or maximum of a function such as the potential energy. In this paper, we consider several well-known molecular conformation problems to which we apply a new method of deterministic global optimization called the cutting angle method. We demonstrate that this method is competitive with other global optimization techniques for these molecular conformation problems.

The salt wedge position in a bar-blocked estuary subject to pulsed inflows

A series of laboratory experiments were carried out to investigate the response of a bar-blocked, saltwedge estuary to the imposition of both steady freshwater inflows and transient inflows that simulate storm events in the catchment area or the regular water releases from upstream reservoirs. The trapped salt water forms a wedge within the estuary, which migrates downstream under the influence of the freshwater inflow. The experiments show that the wedge migration occurs in two stages, namely (i) an initial phase characterized by intense shear-induced mixing at the nose of the wedge, followed by (ii) a relatively quiescent phase with significantly reduced mixing in which the wedge migrates more slowly downstream.

Provided that the transition time t_T between these two regimes satisfies t_T>g′h⁴L/q³α, as was the case for all our experiments and is likely to be the case for most estuaries, then the transition occurs at time t_T=1.2(gα³L⁶/g′³q²)^1/6, where g′=gΔρ/ρ0 is the reduced gravity, g the acceleration due to gravity, Δρ the density excess of the saline water over the density ρ₀ of the freshwater, q the river inflow rate per unit width, and L and α are the length and bottom slope of the estuary, respectively.

A simple model, based on conversion of the kinetic energy of the freshwater inflow into potential energy to mix the salt layer, was developed to predict the displacement xw over time t of the saltwedge nose from its initial position. For continuous inflows subject to t<t_T, the model predicts the saltwedge displacement as x_w/h=1.1 (t/τ)^1/3, where the normalizing length and time scales are h=(q²/g)^1/3 and τ=g′α²h4L/q³, respectively. For continuous inflows subject to t>tT, the model predicts the displacement as xw/h=0.45N1/6(t/τ)1/6/α, where N=q²/g′h²L is a non-dimensional number for the problem. This model shows very good agreement with the experiments. For repeated, pulsed discharges subject to t<t_T, the saltwedge displacement is given by (xw/h)3−(x0/h)(xw/h)2=1.3t/τ, where x₀ is the initial displacement following one discharge event but prior to the next event. For pulsed discharges subject to t>t_T, the displacement is given by (xw/h)⁶−(x0/h)(xw/h)⁵=0.008N(t/τ)/α⁶. This model shows very good agreement with the experiments for the initial discharge event but does systematically underestimate the wedge position for the subsequent pulses. However, the positional error is less than 15%.

Understanding ring strain and ring flexibility in six - and eight-membered cyclic organometallic group 14 oxides

A simple model was developed for the approximation of ring strain energies of homo- and heterometallic, six- and eight-membered cyclic organometallic group 14 oxides and the degree of puckering of their ring conformations. The conformational energy of a ring is modelled as the sum of its angular strain components. The bending potential energy functions for the various endocyclic M–O–M′ and O–M–O linkages (M, M′=Si, Ge, Sn) were calculated at the B3LYP/(v)TZ level of theory using H3MOM′H3 and H2M(OH)2 as model compounds. For the six-membered rings, the minimum total angular contribution to ring strain, ERSGmin was calculated to decrease in the order: cyclo-(H2SiO)3 (13.0 kJ mol−1)>cyclo-H2Sn(OSiH2)2O (7.0 kJ mol−1)>cyclo-H2Ge(OSiH2)2O (4.9 kJ mol−1)>cyclo-H2Si(OSnH2)2O (3.4 kJ mol−1)>cyclo-(H2SnO)3 (1.7 kJ mol−1)>cyclo-H2Si(OGeH2)2O (0.8 kJ mol−1)≈cyclo-H2Ge(OSnH2)2O (0.7 kJ mol−1)>cyclo-H2Sn(OGeH2)2O (0.1 kJ mol−1)≈cyclo-(H2GeO)3 (0 kJ mol−1). All of the six-membered rings were predicted to adopt (nearly) planar conformations (a=0.996<a<1). By contrast, all eight-membered rings were predicted to adopt strainless, but puckered conformations. The degree of puckering was predicted to increase in the order: cyclo-(H2SiO)4 (a=0.983)<cyclo-H2Sn(OSiH2O)2SiH2 (a=0.959)<cyclo-(H2SiO)2(H2SnO)2 (a=0.942)< cyclo-H2Si(OSnH2O)2SiH2 (a=0.935)<cyclo-(H2SnO)4 (a=0.916)<cyclo-(H2GeO)4 (a=0.885). The differences in ring strain and the degree of puckering were linked to the different electronegativities of Si, Ge and Sn. The results obtained are consistent with experimental ring strain energies; reactivities towards ring opening polymerizations or ring expansion reactions and observed ring conformations of cyclic organometallic group 14 oxides.

Mode II delamination toughness of Z-pinned laminates

Mode II delamination toughness of z-pin reinforced composite laminates is investigated using finite element (FE) method. The z-pin pullout process is simulated by the deformation and breakage of non-linear springs. A critical shear stress criterion based on linear elastic fracture mechanics is used to simulate crack growth in an end-notched-flexure (ENF) beam made of z-pinned laminates. The mode II toughness is quantified by the potential energy release rate calculated using the contour integral method. This FE model is verified for an unpinned ENF composite beam. Numerical results obtained indicate that z-pins can significantly increase the mode II delamination toughness of composite laminate. The effects of design variables on the toughness enhancement of z-pinned laminates are also studied, which provides an important technological base and useful data to optimize and improve the z-pinning technique.

Challenges of continuous global optimization in molecular structure prediction

The molecular geometry, the three dimensional arrangement of atoms in space, is a major factor determining the properties and reactivity of molecules, biomolecules and macromolecules. Computation of stable molecular conformations can be done by locating minima on the potential energy surface (PES). This is a very challenging global optimization problem because of extremely large numbers of shallow local minima and complicated landscape of PES. This paper illustrates the mathematical and computational challenges on one important instance of the problem, computation of molecular geometry of oligopeptides, and proposes the use of the Extended Cutting Angle Method (ECAM) to solve this problem.

ECAM is a deterministic global optimization technique, which computes tight lower bounds on the values of the objective function and fathoms those part of the domain where the global minimum cannot reside. As with any domain partitioning scheme, its challenge is an extremely large partition of the domain required for accurate lower bounds. We address this challenge by providing an efficient combinatorial algorithm for calculating the lower bounds, and by combining ECAM with a local optimization method, while preserving the deterministic character of ECAM.

Effect of environmental flows on deep, anoxic pools

A series of field surveys were carried out on two permanent pools of the upper Glenelg River in SW Victoria, Australia. One was representative of the wider and deeper pools while the other was representative of the more-narrow and shallower pools. Both pools showed a typical seasonal cycle of warm, brackish, oxygen-poor, summer conditions and cool, oxygen-rich, low-salinity, winter conditions. The summer salinity increases were larger than expected, suggesting possible saline groundwater inflow from unidentified springs. Both pools contained anoxic water in their deeper sections but this was permanent only in the deeper pool. A simple model of the flushing rate of such anoxic pools subject to flows, such as environmental flow releases, was developed, based on an energy balance between the potential energy required to lift the anoxic layer and the kinetic energy derived from the river flow. The results were tested against and in agreement with the field measurements. The model also suggests that the anoxic layers are resilient to all but the largest environmental flows.

Oxygen-dependence of metabolic rate in the muscles of craniates

We present evidence that oxygen consumption (V_O2 ) is oxygen partial pressure (P_O2) dependent in striated muscles and P_O2 -independent in the vasculature in representatives of three craniate taxa: two teleost fish, a hagfish and a rat. Blood vessel V_O2 displayed varying degrees of independence in a P_O2 range of 15–95 mmHg, while V_O2 by striated muscle tissue slices from all species related linearly to P_O2 between 0 and 125 mmHg, despite V_O2 rates varying greatly between species and muscle type. In salmon red muscle, lactate concentrations fell in slices incubated at a P_O2 of either 30 or 100 mmHg, suggesting aerobic rather than anaerobic metabolism. Consistent with this finding, potential energy, a proxy of ATP turnover, was P_O2 -dependent. Our data suggest that the reduction in V_O2 with falling P_O2 results in a decrease in ATP demand, suggesting that the hypoxic signal is sensed and cellular changes effected. Viability and diffusion limitation of the preparations were investigated using salmon cardiac and skeletal muscles. Following the initial P_O2 depletion, reoxygenation of the Ringer bathing salmon cardiac muscle resulted in V_O2^s that was unchanged from the first run. V_O2 increased in all muscles uncoupled with p-trifluoromethoxylphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP). Mitochondrial succinate dehydrogenase activity, quantified by reduction of 3-(4,5-dimethylthiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, was constant over the course of the experiment. These three findings indicate that the tissues remained viable over time and ruled out diffusion-limitation as a constraint on V_O2.

Effects of sea temperature and stratification changes on seabird breeding success

As apex predators in marine ecosystems, seabirds may primarily experience climate change impacts indirectly, via changes to their food webs. Observed seabird population declines have been linked to climate-driven oceanographic and food web changes. However, relationships have often been derived from relatively few colonies and consider only sea surface temperature (SST), so important drivers, and spatial variation in drivers, could remain undetected. Further, ex - plicit climate change projections have rarely been made, so longer-term risks remain unclear. Here, we use tracking data to estimate foraging areas for 11 black-legged kittiwake Rissa tridac - ty la colonies in the UK and Ireland, thus reducing reliance on single colonies and allowing calculation of colony-specific oceanographic conditions. We use mixed models to consider how SST, the potential energy anomaly (indicating density stratification strength) and the timing of seasonal stratification influence kittiwake productivity. Across all colonies, higher breeding success was associated with weaker stratification before breeding and lower SSTs during the breeding season. Eight colonies with sufficient data were modelled individually: higher productivity was associated with later stratification at 3 colonies, weaker stratification at 2, and lower SSTs at one, whilst 2 colonies showed no significant relationships. Hence, key drivers of productivity varied among colonies. Climate change projections, made using fitted models, indicated that breeding success could decline by 21 to 43% between 1961-90 and 2070-99. Climate change therefore poses a longer-term threat to kittiwakes, but as this will be mediated via availability of key prey species, other marine apex predators could also face similar threats.

Rapid surface functionalization of carbon fibres using microwave irradiation in an ionic liquid

The modification of carbon fibre surfaces has been achieved using a novel combination of low power microwave irradiation (20 W) in both an ionic liquid (1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide) and an organic solvent (1,2-dichlorobenzene). The use of the ionic liquid was superior to the organic solvent in this application, resulting in a higher density of surface grafted material. As a consequence, carbon fibres treated in the ionic liquid displayed improved interfacial adhesion in the composite material (+28% relative to untreated fibres) compared to those treated in organic solvent (+18%). The methodology presented herein can be easily scaled up to industrially relevant quantities and represent a drastic reduction in both reaction time (30 min from 24 h) and energy consumption, compared to previously reported procedures. This work opens the door to potential energy and time saving strategies which can be applied to carbon fibre manufacture for high performance carbon fibre reinforced composites.

Cross-strand disulfides in the non-hydrogen bonding site of antiparallel β-sheet (aCSDns): poised for biological switching

Forbidden disulfides are stressed disulfides found in recognisable protein contexts previously defined as structurally forbidden. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. The meta-stability of forbidden disulfides makes them likely candidates as redox switches. Here we mined the Protein Data Bank for examples of the most common forbidden disulfide, the aCSDn. This is a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen bonded moieties are directed away from each other. We grouped these aCSDns into homologous clusters and performed an extensive physicochemical and informatic analysis of the examples found. We estimated their torsional energies using quantum chemical calculations and studied differences between the preferred conformations of the computational model and disulfides found in solved protein structures to understand the interaction between the forces imposed by the disulfide linkage and typical constraints of the surrounding β-sheet. In particular, we assessed the twisting, shearing and buckling of aCSDn-containing β-sheets, as well as the structural and energetic relaxation when hydrogen bonds in the motif are broken. We show the strong preference of aCSDns for the right-handed staple conformation likely arises from its compatibility with the twist, shear and Cα separation of canonical β-sheet. The disulfide can be accommodated with minimal distortion of the sheet, with almost all the strain present as torsional strain within the disulfide itself. For each aCSDn cluster, we summarise the structural and strain data, taxonomic conservation and any evidence of redox activity. aCSDns are known substrates of thioredoxin-like enzymes. This, together with their meta-stability, means they are ideally suited to biological switching roles and are likely to play important roles in the molecular pathways of oxidative stress.

The potential to reduce the embodied energy in construction through the use of renewable materials

The threat of dangerous levels of global warming demand that we significantly reduce carbon emissions over the coming decades. Globally, carbon emissions from all energy end-uses in buildings in 2004 were estimated to be 8.6 Gt CO2 or almost one quarter of total CO2 emissions (IPCC 2007). In Australia, nearly ten per cent of greenhouse gases come from the residential sector (DCCEE 2012). However, it is not merely the operation of the buildings that contributes to their CO2 emissions, but the energy used over their entire life cycle. Research has demonstrated that the embodied energy of the construction materials used in a building can sometimes equal the operational energy over the building’s entire lifetime (Crawford 2011). Therefore the materials used in construction need to be carefully considered. Conventional building materials not only represent high levels of embodied energy but also use resources that are finite and are being depleted. Renewable building materials are those materials that can be regenerated quickly enough to remove the threat of depletion and in theory their production could be carbon-neutral. To assess the potential for renewable building materials to reduce the embodied energy content of residential construction, the embodied energy of a small residential building has been determined. Wherever possible, the conventional construction materials were then replaced by commercially-available renewable building materials. The embodied energy of the building was then recalculated. The analysis showed that the embodied energy of the building could be reduced from 7.5 GJ per m2 to 5.4 GJ per m2 i.e. by 28%. The commercial availability of renewable materials, however, was a limiting factor and indicated that the industry is not yet well positioned to embrace this strategy to reduce embodied energy of construction. While some conventional building materials could readily be replaced, in many instances a renewable substitute could not be found.