Ocean acidification affects marine chemical communication by changing structure and function of peptide signalling molecules.

Ocean acidification is a global challenge that faces marine organisms in the near future with a predicted rapid drop in pH of up to 0.4 units by the end of this century. Effects of the change in ocean carbon chemistry and pH on the development, growth and fitness of marine animals are well documented. Recent evidence also suggests that a range of chemically mediated behaviours and interactions in marine fish and invertebrates will be affected. Marine animals use chemical cues, for example, to detect predators, for settlement, homing and reproduction. But, while effects of high CO2 conditions on these behaviours are described across many species, little is known about the underlying mechanisms, particularly in invertebrates. Here, we investigate the direct influence of future oceanic pH conditions on the structure and function of three peptide signalling molecules with an interdisciplinary combination of methods. NMR spectroscopy and quantum chemical calculations were used to assess the direct molecular influence of pH on the peptide cues, and we tested the functionality of the cues in different pH conditions using behavioural bioassays with shore crabs (Carcinus maenas) as a model system. We found that peptide signalling cues are susceptible to protonation in future pH conditions, which will alter their overall charge. We also show that structure and electrostatic properties important for receptor binding differ significantly between the peptide forms present today and the protonated signalling peptides likely to be dominating in future oceans. The bioassays suggest an impaired functionality of the signalling peptides at low pH. Physiological changes due to high CO2 conditions were found to play a less significant role in influencing the investigated behaviour. From our results, we conclude that the change of charge, structure and consequently function of signalling molecules presents one possible mechanism to explain altered behaviour under future oceanic pH conditions.

Spore exines increase vitamin D clinical bioavailability by mucoadhesion and bile triggered release.

Sporopollenin exine capsules (SpECs) are microcapsules derived from the outer shells (exines) of plant spore and pollen grains. This work reports the first clinical study on healthy volunteers to show enhanced bioavailability of vitamin D encapsulated in SpECs from Lycopodium clavatum L. spore grains vs vitamin D alone, and the first evidence (in vitro, ex vivo and in vivo) of mechanisms to account for the enhancement and release of the active in the small intestine. Evidence for mucoadhesion of the SpECs contributing to the mechanism of the enhancement is based on: (i) release profile over time of vitamin D in a double blind cross-over human study showing significant release in the small intestine; (ii) in vivo particle counting data in rat showing preferred retention of SpECs vs synthetic beads; (iii) ex vivo99mTc labelling and counting data using rat small intestine sections showing preferred retention of SpECs vs synthetic beads; (iv) in vitro mucoadhesion data. Triggered release by bile in the small intestine was shown in vitro using solid state NMR and HPLC.

Cholesterol and lipid phases influence the interactions between serotonin receptor agonists and lipid bilayers.

Solid state NMR techniques have been used to investigate the effect that two serotonin receptor 1a agonists (quipazine and LY-165,163) have on the phase behavior of, and interactions within, cholesterol/phosphocholine lipid bilayers. The presence of agonist, and particularly LY-165,163, appears to widen the phase transitions, an effect that is much more pronounced in the presence of cholesterol. It was found that both agonists locate close to the cholesterol, and their interactions with the lipids are modulated by the lipid phases. As the membrane condenses into mixed liquid-ordered/disordered phases, quipazine is pushed up toward the surface of the bilayer, whereas LY-165,163 moves deeper into the lipid chain region. In light of our results, we discuss the role of lipid/drug interactions on drug efficacy.

Dynamic nuclear polarization-enhanced solid-state NMR of a 13C-labeled signal peptide bound to lipid-reconstituted Sec translocon.

Dynamic nuclear polarization (DNP) has made it possible to record 2D double-quantum-filtered (DQF) solid-state NMR (ssNMR) spectra of a signal peptide bound to a lipid-reconstituted SecYEG translocon complex. The small quantity of peptide in the sample (~40 nmol) normally prohibits multidimensional ssNMR experiments. Such small amounts are not the exception, because for samples involving membrane proteins, most of the limited sample space is occupied by lipids. As a consequence, a conventional 2D DQF ssNMR spectrum with the sample used here would require many weeks if not months of measurement time. With the help of DNP, however, we were able to acquire such a 2D spectrum within 20 h. This development opens up new possibilities for membrane protein studies, particularly in the exploitation of high-resolution spectroscopy and the assignment of individual amino acid signals, in this case for a signal peptide bound to the translocon complex.

Citizen-led sampling to monitor phosphate levels in freshwater environments using a simple paper microfluidic device.

Contamination of waterways is of increasing concern, with recent studies demonstrating elevated levels of antibiotics, antidepressants, household, agricultural and industrial chemicals in freshwater systems. Thus, there is a growing demand for methods to rapidly and conveniently monitor contaminants in waterways. Here we demonstrate how a combination of paper microfluidic devices and handheld mobile technology can be used by citizen scientists to carry out a sustained water monitoring campaign. We have developed a paper-based analytical device and a 3 minute sampling workflow that requires no more than a container, a test device and a smartphone app. The contaminant measured in these pilots are phosphates, detectable down to 3 mg L-1. Together these allow volunteers to successfully carry out cost-effective, high frequency, phosphate monitoring over an extended geographies and periods.

Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence.

Efflux pumps of the small multidrug resistance family bind cationic, lipophilic antibiotics and transport them across the membrane in exchange for protons. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation, and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. We show the existence of such a substrate-transporter complex for the EmrE homolog Mycobacterium tuberculosis TBsmr and its substrate ethidium bromide. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. Sample illumination generates a DeltapH, which results in enhanced ethidium fluorescence intensity, which is abolished when DeltapH or DeltaPsi is collapsed or when the essential residue Glu-13 in TBsmr is exchanged with Ala. This observation shows the formation of a pH-dependent, transient substrate-protein complex between binding and release of ethidium. We have further characterized this state by determining the K(d), by inhibiting ethidium transport through titration with nonfluorescent substrate and by fluorescence anisotropy measurements. Our findings support a model with a single occluded intermediate state in which the substrate is highly immobile.

Influence of Solvent Representation on Nuclear Shielding Calculations of Protonation States of Small Biological Molecules.

In this study, we assess the influence of solvation on the accuracy and reliability of isotropic nuclear magnetic shielding calculations for amino acids in comparison to experimental data. We focus particularly on the performance of solvation methods for different protonation states, as biological molecules occur almost exclusively in aqueous solution and are subject to protonation with pH. We identify significant shortcomings of current implicit solvent models and present a hybrid solvation approach that improves agreement with experimental data by taking into account the presence of direct interactions between amino acid protonation state and water molecules.

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding.

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.

Investigating transport proteins by solid state NMR.

Transporters form an interesting and complex class of membrane proteins. Many of them are potential drug targets due to their role in translocation of ions, small molecules and peptides across the membrane or due to their role in multidrug resistance. Hence elucidating their structure and mechanism is of great importance and may lead to a host of new drugs and methods to alter or inhibit their function. Solid state NMR is an emerging technique for investigating transport proteins. Along with other biochemical and biophysical techniques solid state NMR can provide data on drug binding, protein dynamics and structure at the interface between structural biology and functional analysis. Here, we review solid state NMR applications to primary active and secondary transporters involved in translocation of small molecules. We discuss current experimental limitations and give an overall perspective on how the technique may be used to address some pertinent questions relevant to transporters.

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer.

The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.

Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.

The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles.

Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.

The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition.

The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.

Chelator free gallium-68 radiolabelling of silica coated iron oxide nanorods via surface interactions.

The commercial availability of combined magnetic resonance imaging (MRI)/positron emission tomography (PET) scanners for clinical use has increased demand for easily prepared agents which offer signal or contrast in both modalities. Herein we describe a new class of silica coated iron-oxide nanorods (NRs) coated with polyethylene glycol (PEG) and/or a tetraazamacrocyclic chelator (DO3A). Studies of the coated NRs validate their composition and confirm their properties as in vivo T2 MRI contrast agents. Radiolabelling studies with the positron emitting radioisotope gallium-68 (t1/2 = 68 min) demonstrate that, in the presence of the silica coating, the macrocyclic chelator was not required for preparation of highly stable radiometal-NR constructs. In vivo PET-CT and MR imaging studies show the expected high liver uptake of gallium-68 radiolabelled nanorods with no significant release of gallium-68 metal ions, validating our innovation to provide a novel simple method for labelling of iron oxide NRs with a radiometal in the absence of a chelating unit that can be used for high sensitivity liver imaging.

Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.

The ABC transporter LmrA in Lactococcus lactis confers resistance to a wide range of antibiotics and cytotoxic drugs and is a functional homologue of P-glycoprotein. Recently, solid-state NMR methods have shown potential for structural- and non-perturbing, site directed functional studies. These experiments require isotopic labelling of selected sites. We have developed a strategy to produce large quantities of selectively labelled LmrA reconstituted at a high density in lipid membranes. This makes the 64 kDa integral membrane protein LmrA and therefore the ABC transporter superfamily accessible to NMR analysis.

Whiter, brighter, and more stable cellulose paper coated with TiO2 /SiO2 core/shell nanoparticles using a layer-by-layer approach.

To inhibit the photocatalytic degradation of organic material supports induced by small titania (TiO2 ) nanoparticles, four kinds of TiO2 nanoparticles, that is, commercial P25-TiO2 , commercial rutile phase TiO2 , rutile TiO2 nanorods and rutile TiO2 spheres, prepared from TiCl4 , were coated with a thin, but dense, coating of silica (SiO2 ) using a conventional sol-gel technique to form TiO2 /SiO2 core/shell nanoparticles. These core/shell particles were deposited and fixed as a very thin coating onto the surface of cellulose paper samples by a wet-chemistry polyelectrolyte layer-by-layer approach. The TiO2 /SiO2 nanocoated paper samples exhibit higher whiteness and brightness and greater stability to UV-bleaching than comparable samples of blank paper. There are many potential applications for this green chemistry approach to protect cellulosic fibres from UV-bleaching in sunlight and to improve their whiteness and brightness.

Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking.

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers.

MRI contrast agent delivery using spore capsules: controlled release in blood plasma.

The exine coatings of spores can be used to encapsulate drug molecules. We have demonstrated that these microcapsules can be filled with a commercial gadolinium(III) MRI contrast agent (in this proof of concept study Gd-DTPA-BMA was used) which is slowly released in plasma due to enzymatic digestion of the capsule.

Location and orientation of serotonin receptor 1a agonists in model and complex lipid membranes.

Magic angle spinning (MAS) NMR has been used to investigate the location and orientation of five serotonin receptor 1a agonists (serotonin, buspirone, quipazine, 8-OH-DPAT, and LY-163,165) in single component model lipid and brain lipid membranes. The agonist locations are probed by monitoring changes in the lipid proton chemical shifts and by MAS-assisted nuclear Overhauser enhancement spectroscopy, which indicates the orientation of the agonists with respect to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids. In the single component bilayer, the membrane agonists are found predominantly in the top of the hydrophobic chain or in the glycerol region of the membrane. Most of the agonists orient approximately parallel to the membrane plane, with the exception of quipazine, whose piperazine ring is found in the glycerol region, whereas its benzene ring is located within the lipid hydrophobic chain. The location of the agonist in brain lipid membranes is similar to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers; however, many of the agonists appear to locate close to the cholesterol in the membrane in preference to the phospholipids.

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR.

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin.