Correction: On demand modulation of lipid composition in an individual bilayer.

Correction for 'On demand modulation of lipid composition in an individual bilayer' by John S. H. Danial et al., Soft Matter, 2017, 13, 1788-1793.

On demand modulation of lipid composition in an individual bilayer.

Changes in local lipid composition are thought to play a key role in regulating many complex cellular processes. By studying lipid organization in artificial lipid bilayers the physical principles underlying these process can be studied in detail. However, such in vitro measurements are often hindered by heterogeneities in the lipid composition of individual bilayers prepared by current bulk methods. Here, the lipid composition of an individual droplet interface bilayer is varied by lipid titration into the bilayer from the oil phase in a microfluidic device. Control of lipid composition allows the reversible switching between single- and two-phase regions and sampling of specific lipid compositions in an individual bilayer. This method enables controlled modulation of composition-sensitive processes in a single lipid membrane.

Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers.

Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in individual pores.

Constructing droplet interface bilayers from the contact of aqueous droplets in oil.

We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.

Imaging multiple conductance states in an alamethicin pore.

Alamethicin is the archetypal antimicrobial pore-forming peptide. Although the peptide has long been known to form pores of characteristic conductances in lipid membranes, the precise nature of these pores is not known. Simultaneous calcium-flux imaging and single-channel recording in a droplet interface bilayer allowed us to directly attribute multiple conductance states to a single point diffusing in the bilayer.

Rapid assembly of a multimeric membrane protein pore.

We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in <5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.

Lucky imaging: improved localization accuracy for single molecule imaging.

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells.

Simultaneous measurement of ionic current and fluorescence from single protein pores.

The ability to simultaneously monitor both the ionic current and fluorescence from membrane channels and pores has the potential to link structural changes with function in such proteins. We present a new method for simultaneously measuring single-channel electrical currents and fluorescence from membrane proteins by using water-in-oil droplet bilayers. We demonstrate the simultaneous fluorescence and electrical detection of stochastic blocking by cyclodextrin in multiple staphylococcal alpha-hemolysin pores. The combined fluorescence signal from individual pores exhibits the same sequence of blocking events as the total current recording, showing that the two signals from each pore are correlated.

Quantitative (upsilon, N, Ka) product state distributions near the triplet threshold for the reaction H2CO --> H + HCO measured by Rydberg tagging and laser-induced fluorescence.

In this paper, we report quantitative product state distributions for the photolysis of H2CO --> H + HCO in the triplet threshold region, specifically for several rotational states in the 2(2)4(3) and 2(3)4(1) H2CO vibrational states that lie in this region. We have combined the strengths of two complementary techniques, laser-induced fluorescence for fine resolution and H atom Rydberg tagging for the overall distribution, to quantify the upsilon, N, and Ka distributions of the HCO photofragment formed via the singlet and triplet dissociation mechanisms. Both techniques are in quantitative agreement where they overlap and provide calibration or benchmarks that permit extension of the results beyond that possible by each technique on its own. In general agreement with previous studies, broad N and Ka distributions are attributed to reaction on the S0 surface, while narrower distributions are associated with reaction on T1. The broad N and Ka distributions are modeled well by phase space theory. The narrower N and Ka distributions are in good agreement with previous quasi-classical trajectory calculations on the T1 surface. The two techniques are combined to provide quantitative vibrational populations for each initial H2CO vibrational state. For dissociation via the 2(3)4(1) state, the average product vibrational energy (15% of E(avail)) was found to be about half of the rotational energy (30% of E(avail)), independent of the initial H2CO rotational state, irrespective of the singlet or triplet mechanism. For dissociation via the 2(2)4(3) state, the rotational excitation remained about 30% of E(avail), but the vibrational excitation was reduced.

Asymmetric droplet interface bilayers.

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.

Droplet interface bilayers.

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

State-selective photodissociation dynamics of formaldehyde: near threshold studies of the H+HCO product channel.

The laser-induced photodissociation of formaldehyde in the wavelength range 309

Near-UV photolysis of substituted phenols, I: 4-fluoro-, 4-chloro- and 4-bromophenol.

The experimental techniques of H (Rydberg) atom photofragment translational spectroscopy and resonance-enhanced multiphoton ionisation time-of-flight spectroscopy have been used to investigate the dynamics of H atom loss processes from gas phase 4-fluorophenol (4-FPhOH), 4-chlorophenol (4-ClPhOH) and 4-bromophenol (4-BrPhOH) molecules, following excitation at many wavelengths, lambda(phot), in the range between their respective S(1)-S(0) origins (284.768 nm, 287.265 nm and 287.409 nm) and 216 nm. Many of the Total Kinetic Energy Release (TKER) spectra obtained from photolysis of 4-FPhOH show structure, the analysis of which reveals striking parallels with that reported previously for photolysis of bare phenol (M. G. D. Nix, A. L. Devine, B. Cronin, R. N. Dixon and M. N. R. Ashfold, J. Chem. Phys., 2006, 125, 133318). The data demonstrates the importance of O-H bond fission, and that the resulting 4-FPhO co-fragments are formed in a select fraction of their available vibrational state density. All spectra recorded at lambda(phot)> or = 238 nm show a feature centred at TKER approximately 5500 cm(-1). These H atom fragments show no recoil anisotropy, and are rationalised in terms of initial S(1)<-- S(0) (pi* <--pi) excitation and subsequent dissociation via two successive radiationless transitions: internal conversion to ground (S(0)) state levels carrying sufficient O-H stretch vibrational energy to allow efficient transfer to (and round) the Conical Intersection (CI) between the S(0) and S(2)((1)pi sigma*) Potential Energy Surfaces (PESs) at larger R(O-H), en route to H atoms and ground state 4-FPhO products. The vibrational energy disposal in the 4-FPhO products indicates that parent mode nu(16a) promotes non-adiabatic coupling at the S(0)/S(2) CI. Spectra recorded at lambda(phot)< or = 238 nm reveal a faster (but still isotropic) distribution of recoiling H atoms, centred at TKER approximately 12 000 cm(-1), attributable to H + 4-FPhO products formed when the optically excited (1)pi pi* molecules couple directly with the (1)pi sigma* PES. Parent mode nu(16b) is identified as the dominant coupling mode at the S(1)((1)pi pi*)/S(2)((1)pi sigma*) CI, and the resulting 4-FPhO radical co-fragments display progressions in nu(18b) (the C-O in-plane wagging mode) and nu(7a) (an in-plane ring breathing mode involving significant C-O stretching motion). Analysis of all structured TKER spectra yields a C-F bond dissociation energy: D(0)(H-OC(6)H(4)F) = 29 370 +/- 50 cm(-1). The photodissociation of 4-ClPhOH shows many similarities, though the 4-ClPhO products formed together with faster H atoms at shorter wavelengths (lambda(phot)< or = 238 nm, by coupling through the S(1)/S(2) CI) show activity in an alternative ring breathing mode (nu(19a) rather than nu(7a)). Spectral analysis yields D(0)(H-OC(6)H(4)Cl) = 29 520 +/- 50 cm(-1). H atom formation via O-H bond fission is (at best) a very minor channel in the photolysis of 4-BrPhOH at all wavelengths investigated. Time-dependent density functional theory calculations suggest that this low H atom yield is because of competition from the alternative C-Br bond fission channel, and that the analogous C-Cl bond fission may be responsible for the weakness of the one photon-induced H atom signals observed when photolysing 4-ClPhOH at longer wavelengths.

Ultraviolet photolysis of adenine: dissociation via the 1pi sigma* state.

High resolution total kinetic energy release (TKER) spectra of the H atom fragments resulting from photodissociation of jet-cooled adenine molecules at 17 wavelengths in the range 280>lambda(phot)>214 nm are reported. TKER spectra obtained at lambda(phot)>233 nm display broad, isotropic profiles that peak at low TKER ( approximately 1800 cm(-1)) and are largely insensitive to the choice of excitation wavelength. The bulk of these products is attributed to unintended multiphoton dissociation processes. TKER spectra recorded at lambda(phot)

High resolution photofragment translational spectroscopy studies of the near ultraviolet photolysis of imidazole.

The fragmentation dynamics of imidazole molecules following excitation at 193.3 nm and at many wavelengths in the range of 210< or =lambda(phot)< or =240 nm have been investigated by H Rydberg atom photofragment translational spectroscopy. Long wavelength excitation within this range results in population of the 1 (1)A(")((1)pisigma(*)) excited state, but the 2 (1)A(')<--X (1)A(')(pi(*)<--pi) transition becomes the dominant absorption once lambda(phot)< or =220 nm. The measured energy disposals show parallels with those found in recent studies of the UV photolysis of pyrrole [Cronin et al., Phys Chem. Chem. Phys. 6, 5031 (2004)]. The total kinetic energy release (TKER) spectra display a "fast" feature, centred at TKER approximately 9200 cm(-1). The analysis of the structure evident in the fast feature reveals the selective population of specific in-plane stretching vibrational levels of the imidazolyl cofragment; these fragments are deduced to carry only modest amounts of rotational excitation. Comparison with calculated normal mode vibrational frequencies allows the assignment of the populated levels and a precise determination of the N-H bond strength in imidazole: D(0)=33,240+/-40 cm(-1). The observed energy disposal can be rationalized using Franck-Condon arguments, assuming that the potential energy surface (PES) for the 1 (1)A(")((1)pisigma(*)) state has a topology similar to that of the corresponding (1)pisigma(*) state of pyrrole. As in pyrrole, photoexcitation populates skeletal motions in the S(1) state (in-plane motions in the present case) that are only weakly coupled to the N-H dissociation coordinate and thus map through into the corresponding product vibrations. A second, "slow" feature is increasingly evident in TKER spectra recorded at shorter lambda(phot). This component, which exhibits no recoil anisotropy, is attributed to H atoms formed by the "statistical" decay of highly vibrationally excited ground state molecules. The form of the TKER spectra observed at short lambda(phot) is rationalized by assuming two possible decay routes for imidazole molecules excited to the 2 (1)A(')((1)pipi(*)) state. One involves fast 2 (1)A(')((1)pipi(*)) right arrow-wavy 1 (1)A(")((1)pisigma(*)) radiationless transfer and subsequent fragmentation on the 1 (1)A(')((1)pisigma(*)) PES, yielding fast H atoms (and imidazolyl cofragments)-reminiscent of behavior seen at longer excitation wavelengths where the 1 (1)A(")((1)pisigma(*)) PES is accessed directly. The second is assumed to involve radiationless transfer to the ground state, most probably by successive 2 (1)A(') right arrow-wavy 1 (1)A(") right arrow-wavy X (1)A(') couplings, mediated by conical intersections between the relevant PESs and the subsequent unimolecular decay of the resulting highly vibrationally excited ground state molecules yielding slow H atoms.

High resolution photofragment translational spectroscopy studies of the near ultraviolet photolysis of phenol.

The fragmentation dynamics of gas phase phenol molecules following excitation at many wavelengths in the range 279.145 > or = lambdaphot > or = 206.00 nm have been investigated by H Rydberg atom photofragment translational spectroscopy. Many of the total kinetic energy release (TKER) spectra so derived show structure, the analysis of which confirms the importance of O-H bond fission and reveals that the resulting phenoxyl cofragments are formed in a very limited subset of their available vibrational state density. Spectra recorded at lambdaphot > or = 248 nm show a feature centered at TKER approximately 6500 cm(-1). These H atom fragments, which show no recoil anisotropy, are rationalized in terms of initial S1<--S0 (pi*<--pi) excitation, and subsequent dissociation via two successive radiationless transitions: internal conversion to ground (S0) state levels carrying sufficient O-H stretch vibrational energy to allow efficient transfer towards, and passage around, the conical intersection (CI) between the S0 and S2(1pisigma*) potential energy surfaces (PESs) at larger R(O-H), en route to ground state phenoxyl products. The observed phenoxyl product vibrations indicate that parent modes nu16a and nu11 can both promote nonadiabatic coupling in the vicinity of the S0S2 CI. Spectra recorded at lambdaphot < or = 248 nm reveal a faster, anisotropic distribution of recoiling H atoms, centered at TKER approximately 12,000 cm(-1). These we attribute to H+phenoxyl products formed by direct coupling between the optically excited S1(1pi pi*) and repulsive S2(1pi sigma*) PESs. Parent mode nu16b is identified as the dominant coupling mode at the S1/S2 CI, and the resulting phenoxyl radical cofragments display a long progression in nu18b, the C-O in-plane wagging mode. Analysis of all structured TKER spectra yields D0(H-OC6H5) = 30,015 +/- 40 cm(-1). The present findings serve to emphasize two points of wider relevance in contemporary organic photochemistry: (i) The importance of 1) pi sigma* states in the fragmentation of gas phase heteroaromatic hydride molecules, even in cases where the 1pi sigma* state is optically dark. (ii) The probability of observing strikingly mode-specific product formation, even in "indirect" predissociations, if the fragmentation is driven by ultrafast nonadiabatic couplings via CIs between excited (and ground) state PESs.

Absorption cross sections of formaldehyde at wavelengths from 300 to 340 nm at 294 and 245 K.

Absorption cross sections for the A1A2-X1A1 electronic transition of formaldehyde have been measured by ultraviolet (UV) laser absorption spectroscopy in the tropospherically significant wavelength range 300-340 nm, over which HCHO is photochemically active. Absorption cross sections are reported at two temperatures, 294 and 245 K and at a spectral resolution of 0.0035 nm (0.35 cm-1). At this resolution, greater peak absorption cross sections are obtained for many of the sharp spectral features than were previously reported. To simulate atmospheric conditions in the troposphere, the effects of adding a pressure of nitrogen of up to 500 Torr and of reduced sample temperature were investigated. The overall magnitudes of peak absorption cross sections are largely unaffected by the added pressure of nitrogen, but a modest degree of pressure broadening (0.2-0.3 cm-1 atm-1) is evident in the line shapes. Computer simulations of spectra have been optimized by comparison with wavelength-dependent formaldehyde absorption cross sections for each major vibronic band in the chosen wavelength range. Experimental and computer simulated spectra at 294 and 245 K are compared to test the reliability of the computer simulations for quantification of the effects of temperature on absorption cross sections. All experimental absorption cross section data and tables of input parameters for spectral simulations are available as Supporting Information.

Near ultraviolet photolysis of deuterated pyrrole.

High resolution time-of-flight (TOF) measurements of the D atom fragments arising in the near ultraviolet (UV) photodissociation of deuterated pyrrole are reported. Structures evident in the measured TOF spectra are all interpretable in terms of N-D bond fission, and population of selected vibrational states of the pyrrolyl-d(4) co-fragment -- thereby clarifying previous uncertainties regarding the branching into different vibronic states of the pyrrolyl radical following UV excitation of pyrrole.

High resolution photofragment translational spectroscopy studies of the near ultraviolet photolysis of 2,5-dimethylpyrrole.

The photodissociation dynamics of 2,5-dimethylpyrrole (2,5-DMP) has been investigated following excitation at 193.3 nm and at many near ultraviolet (UV) wavelengths in the range 244 < lambda(phot) < 282 nm using H Rydberg atom photofragment translational spectroscopy (PTS). Complementary UV absorption and, at the longest excitation wavelengths, one photon resonant multiphoton ionisation spectra of 2,5-DMP are reported also; analysis of the latter highlights the role of methyl torsional motions in promoting the parent absorption. The deduced fragmentation dynamics show parallels with that reported recently (B. Cronin, M. G. D. Nix, R. H. Qadiri and M. N. R. Ashfold, Phys. Chem. Chem. Phys., 2004, 6, 5031) for the bare pyrrole molecule. Excitation at the longer wavelengths leads to (vibronically induced) population of the 1(1)A(2)(pisigma*) excited state of 2,5-DMP, but once lambda(phot) decreases to approximately 250 nm stronger, dipole allowed transitions start to become apparent in the parent absorption. All total kinetic energy release (TKER) spectra of the H + 2,5-dimethylpyrrolyl (2,5-DMPyl) fragments measured at lambda(phot)> or=244 nm show a structured fast component, many of which are dominated by a peak with TKER approximately 5100 cm(-1); analysis of this structure reveals lambda(phot) dependent population of selected vibrational levels of 2,5-DMPyl, and enables determination of the N-H bond strength in 2,5-DMP: D(0) = 30 530 +/- 100 cm(-1). Two classes of behaviour are proposed to account for details of the observed energy partitioning. Both assume that N-H bond fission involves passage over (or tunnelling through) a small exit channel barrier on the 1(1)A(2) potential energy surface, but differ according to the vibrational energy content of the photo-prepared molecules. Specific parent out-of-plane skeletal modes that promote the 1(1)A(2)-X(1)A(1) absorption appear to evolve adiabatically into the corresponding vibrations of the 2,5-DMPyl products. Methyl torsions can also promote the 1(1)A(2)<-- X(1)A(1) absorption in 2,5-DMP, and provide a means of populating a much higher density of excited vibrational levels than in pyrrole. Such excited levels are deduced to dissociate by redistributing the minimum amount of internal energy necessary to overcome the exit channel barrier in the N-H dissociation coordinate. Coupling with the ground state surface via a conical intersection at extended N-H bond lengths is proposed as a further mechanism for modest translational --> vibrational energy transfer within the separating products. The parent absorption cross-section increases considerably at wavelengths approximately 250 nm, and PTS spectra recorded at lambda(phot)< or = 254 nm display a second, unstructured, peak at lower TKER. As in pyrrole, this slower component is attributed to H atoms from the unimolecular decay of highly vibrationally excited ground state molecules formed via radiationless decay from photo-excited states lying above the 1(1)A(2) state.

Photodissociation and photoionization of pyrrole following the multiphoton excitation at 243 and 364.7 nm.

Photoelectron imaging and time of flight mass spectrometry are used to study the multiphoton ionization and dissociation of pyrrole and its cation following excitation at 243 nm and at 364.7 nm. Our results confirm the 8.2 eV ionization potential of pyrrole and the 9.2 eV ionization threshold for forming the 2B1 first excited state of the cation. Prompt photolysis of the N-H bond in neutral pyrrole following one-photon excitation to its 1 1A2 neutral excited state is inferred from analysis of the two-photon photoelectron spectrum recorded at 243 nm, confirming the findings of recent translational spectroscopy studies. Facile dissociation of the pyrrole cation is also observed following excitation at 243 nm; analysis of the fragment cations indicates the operation of a complex dissociation mechanism involving dual bond fission and possible migration of the H atom originally bonded to the nitrogen heteroatom.