Membrane Structure of Aquaporin Observed with Combined Experimental and Theoretical Sum Frequency Generation Spectroscopy.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Membranes Are Decisive for Maximum Freezing Efficiency of Bacterial Ice Nucleators.

Ice-nucleating proteins (INPs) from Pseudomonas syringae are among the most active ice nucleators known, enabling ice formation at temperatures close to the melting point of water. The working mechanisms of INPs remain elusive, but their ice nucleation activity has been proposed to depend on the ability to form large INP aggregates. Here, we provide experimental evidence that INPs alone are not sufficient to achieve maximum freezing efficiency and that intact membranes are critical. Ice nucleation measurements of phospholipids and lipopolysaccharides show that these membrane components are not part of the active nucleation site but rather enable INP assembly. Substantially improved ice nucleation by INP assemblies is observed for deuterated water, indicating stabilization of assemblies by the stronger hydrogen bonds of D2O. Together, these results show that the degree of order/disorder and the assembly size are critically important in determining the extent to which bacterial INPs can facilitate ice nucleation.

Electrostatic Interactions Control the Functionality of Bacterial Ice Nucleators.

Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation more efficiently than any other material. The details of their working mechanism remain elusive, but their high activity has been shown to involve the formation of functional INP aggregates. Here we reveal the importance of electrostatic interactions for the activity of INPs from the bacterium Pseudomonas syringae by combining a high-throughput ice nucleation assay with surface-specific sum-frequency generation spectroscopy. We determined the charge state of nonviable P. syringae as a function of pH by monitoring the degree of alignment of the interfacial water molecules and the corresponding ice nucleation activity. The net charge correlates with the ice nucleation activity of the INP aggregates, which is minimal at the isoelectric point. In contrast, the activity of INP monomers is less affected by pH changes. We conclude that electrostatic interactions play an essential role in the formation of the highly efficient functionally aligned INP aggregates, providing a mechanism for promoting aggregation under conditions of stress that prompt the bacteria to nucleate ice.

Surface-Specific Spectroscopy of Water at a Potentiostatically Controlled Supported Graphene Monolayer.

Knowledge of the structure of interfacial water molecules at electrified solid materials is the first step toward a better understanding of important processes at such surfaces, in, e.g., electrochemistry, atmospheric chemistry, and membrane biophysics. As graphene is an interesting material with multiple potential applications such as in transistors or sensors, we specifically investigate the graphene-water interface. We use sum-frequency generation spectroscopy to investigate the pH- and potential-dependence of the interfacial water structure in contact with a chemical vapor deposited (CVD) grown graphene surface. Our results show that the SFG signal from the interfacial water molecules at the graphene layer is dominated by the underlying substrate and that there are water molecules between the graphene and the (hydrophilic) supporting substrate.

Hydration and Orientation of Carbonyl Groups in Oppositely Charged Lipid Monolayers on Water.

The carbonyl groups of glycerolipid monolayers on water play an important role in the formation of the interfacial hydrogen bond network, which in turn influences the interactions of lipids with, for example, metabolites. As the frequency of the carbonyl absorption band strongly depends on the hydration state of the lipid headgroups, the carbonyl band is a sensitive reporter of changes in the headgroup environment. Here, we use phase-resolved sum frequency generation spectroscopy to obtain information about the orientation and hydration of the carbonyl groups in lipid monolayers. We find that there are two distinct carbonyl moieties in the lipid monolayers, oppositely oriented relative to the surface plane, that experience substantially different hydrogen-bonding environments.

Surface Potential of a Planar Charged Lipid-Water Interface. What Do Vibrating Plate Methods, Second Harmonic and Sum Frequency Measure?

The interfacial electrical potential is an important parameter influencing, for instance, electrochemical reactions and biomolecular interactions at membranes. A deeper understanding of different methods that measure quantities related to the surface potential is thus of great scientific and technological relevance. We use lipid monolayers with varying charge density and thoroughly compare the results of surface potential measurements performed with the vibrating plate capacitor method and second harmonic generation spectroscopy. The two techniques provide very different results as a function of surface charge. Using the molecular information on lipid alkyl chain, lipid headgroup, and interfacial water provided by sum frequency generation spectroscopy, we disentangle the different contributions to the surface potential measured by the different techniques. Our results show that the two distinct approaches are dominated by different molecular moieties and effects. While the shape of the SPOT method response as a function of charge density is dominated by the lipid carbonyl groups, the SHG results contain contributions from the interfacial water molecules, the lipids and hyper-Rayleigh scattering.

Anionic and cationic Hofmeister effects are non-additive for guanidinium salts.

To understand specific ion effects on a molecular level we explore the effect of salts on the rotational mobility of a model amide using dielectric spectroscopy. Based on our previous studies on the effect of strong denaturing anions or cations, here we study the additivity of the anionic and cationic effect. Using salts consisting of denaturing spherical anions and spherical cations we find such salts to affect the amide according to what one expects based on the additive activity of the individual ions. The guanidinium (Gdm+) cation appears to be a notable exception, as our results suggest that GdmI (and accordingly GdmSCN) is less efficient in hindering the rotation of the amide than KI or GdmCl.

Self-referenced ultra-broadband transient terahertz spectroscopy using air-photonics.

Terahertz (THz) air-photonics employs nonlinear interactions of ultrashort laser pulses in air to generate and detect THz pulses. As air is virtually non-dispersive, the optical-THz phase matching condition is automatically met, thus permitting the generation and detection of ultra-broadband THz pulses covering the entire THz spectral range without any gaps. Air-photonics naturally offers unique opportunities for ultra-broadband transient THz spectroscopy, yet many critical challenges inherent to this technique must first be resolved. Here, we present explicit guidelines for ultra-broadband transient THz spectroscopy with air-photonics, including a novel method for self-referenced signal acquisition minimizing the phase error, and the numerically-accurate approach to the transient reflectance data analysis.

Correction: Quantifying transient interactions between amide groups and the guanidinium cation.

Correction for 'Quantifying transient interactions between amide groups and the guanidinium cation' by V. Balos et al., Phys. Chem. Chem. Phys., 2015, 17, 28539-28543.

Quantifying transient interactions between amide groups and the guanidinium cation.

We study the interaction of the guanidinium cation, a widely used protein denaturant, with amide groups, the common structural motif of proteins. Our results provide evidence for direct contact between guanidinium and ∼2 amide groups, but the interaction is transient and weaker than for other cations with high charge-density.

Competing ultrafast energy relaxation pathways in photoexcited graphene.

For most optoelectronic applications of graphene, a thorough understanding of the processes that govern energy relaxation of photoexcited carriers is essential. The ultrafast energy relaxation in graphene occurs through two competing pathways: carrier-carrier scattering, creating an elevated carrier temperature, and optical phonon emission. At present, it is not clear what determines the dominating relaxation pathway. Here we reach a unifying picture of the ultrafast energy relaxation by investigating the terahertz photoconductivity, while varying the Fermi energy, photon energy and fluence over a wide range. We find that sufficiently low fluence (≲4 µJ/cm(2)) in conjunction with sufficiently high Fermi energy (≳0.1 eV) gives rise to energy relaxation that is dominated by carrier-carrier scattering, which leads to efficient carrier heating. Upon increasing the fluence or decreasing the Fermi energy, the carrier heating efficiency decreases, presumably due to energy relaxation that becomes increasingly dominated by phonon emission. Carrier heating through carrier-carrier scattering accounts for the negative photoconductivity for doped graphene observed at terahertz frequencies. We present a simple model that reproduces the data for a wide range of Fermi levels and excitation energies and allows us to qualitatively assess how the branching ratio between the two distinct relaxation pathways depends on excitation fluence and Fermi energy.

Formation of lysozyme oligomers at model cell membranes monitored with sum frequency generation spectroscopy.

A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly α-helix content and an oligomeric (za-mer) state. At pH ≤ 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel ß-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an α-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel ß-sheet structure are found to destabilize lipid membranes.

Structural properties of gp41 fusion peptide at a model membrane interface.

We explore the structure and orientation of N-terminal (23 amino acids) of HIV gp41 envelop protein at the interface of a phospholipid monolayer. Using surface specific sum frequency generation, we probe the response of the Amide I vibrational states of the protein, and compare the experimental results to the modeled response of several secondary structures that have previously been reported in literature. To obtain the modeled response, we derive the line-shape expressions under cumulant expansion within the Brownian oscillator model, and express the intensities of the theoretical response according to the components of the Raman tensors and the infrared transition dipole moments of the relevant Amide I modes, under different orientation angles. This approach enables us to identify one plausible secondary structure among those considered, and allows us to determine the orientation angle, under which the protein inserts into the monolayer. We discuss the relevance of our findings toward understanding the functionality of this polypeptide at the membrane interface.

Determining in situ protein conformation and orientation from the amide-I sum-frequency generation spectrum: theory and experiment.

Vibrational sum-frequency generation (VSFG) spectra of the amide-I band of proteins can give detailed insight into biomolecular processes near membranes. However, interpreting these spectra in terms of the conformation and orientation of a protein can be difficult, especially in the case of complex proteins. Here we present a formalism to calculate the amide-I infrared (IR), Raman, and VSFG spectra based on the protein conformation and orientation distribution. Based on the protein conformation, we set up the amide-I exciton Hamiltonian for the backbone amide modes that generate the linear and nonlinear spectroscopic responses. In this Hamiltonian, we distinguish between nearest-neighbor and non-nearest-neighbor vibrational couplings. To determine nearest-neighbor couplings we use an ab initio 6-31G+(d) B3LYP-calculated map of the coupling as a function of the dihedral angles. The other couplings are estimated using the transition-dipole coupling model. The local-mode frequencies of hydrogen-bonded peptide bonds and of peptide bonds to proline residues are red-shifted. To obtain realistic hydrogen-bond shifts we perform a molecular dynamics simulation in which the protein is solvated by water. As a first application, we measure and calculate the amide-I IR, Raman, and VSFG spectra of cholera toxin B subunit docked to a model cell membrane. To deduce the orientation of the protein with respect to the membrane from the VSFG spectra, we compare the experimental and calculated spectral shapes of single-polarization results, rather than comparing the relative amplitudes of VSFG spectra recorded for different polarization conditions for infrared, visible, and sum-frequency light. We find that the intrinsic uncertainty in the interfacial refractive index--essential to determine the overall amplitude of the VSFG spectra--prohibits a meaningful comparison of the intensities of the different polarization combinations. In contrast, the spectral shape of most of the VSFG spectra is independent of the details of the interfacial refractive index and provides a reliable way of determining molecular interfacial orientation. Specifically, we find that the symmetry axis of the cholera toxin B subunit is oriented at an angle of 6° ± 17° relative to the surface normal of the lipid monolayer, in agreement with 5-fold binding between the toxin's five subunits and the receptor lipids in the membrane.

Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei.

Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30-40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.

Precision waveguide system for measurement of complex permittivity of liquids at frequencies from 60 to 90 GHz.

We describe a variable path length waveguide setup developed to accurately measure the complex dielectric permittivity of liquids. This is achieved by measuring the complex scattering parameter of the liquid in a waveguide section with a vector network analyzer in combination with an E-band frequency converter. The automated measurement procedure allows fast acquisition at closely spaced intervals over the entire measurement bandwidth: 60-90 GHz. The presented technique is an absolute method and as such is not prone to calibration errors. The technique is suited to investigate low-loss as well as high-loss liquids in contrast to similar setups described previously. We present measurements for a high-loss liquid (water), an intermediate-loss sample (ethanol), and for nearly loss-less n-octane. Due to the available phase information, the present data have an improved accuracy in comparison with literature data.

Size-dependent electron transfer from PbSe quantum dots to SnO2 monitored by picosecond Terahertz spectroscopy.

We report the direct and unambiguous determination of electron transfer rates and efficiencies from PbSe quantum dots (QDs) to mesoporous SnO2 films. We monitor the time-dependent electron density within the oxide with picosecond time resolution using Terahertz spectroscopy, following optical excitation of the QDs using a femtosecond laser pulse. QD-oxide electron transfer occurs with efficiencies of ∼2% in our samples under 800 nm pumping with a marked dependence on QD size, ranging from ∼100 ps injection times for the smallest, ∼2 nm diameter QDs, to ∼1 ns time scale for ∼7 nm QDs. The size-dependent electron transfer rates are modeled within the framework of Marcus theory and the implications of the results for device design are discussed.

Anisotropic water reorientation around ions.

We study the reorientation dynamics of water molecules around ions using terahertz dielectric relaxation spectroscopy and polarization-resolved femtosecond infrared pump-probe spectroscopy. The results are discussed in relation to the ion-specific Hofmeister series and the concomitant "structure-making" and "structure-breaking" effects of ions on water. We show that when a dissolved salt consists of a strongly hydrated ion with a weakly hydrated counterion the reorientation of water molecules around the strongly hydrated ion is anisotropic, in the sense that differently charged ions affect reorientation along different molecular axes: cations mainly slow the reorientation dynamics of the water dipole vectors, and anions mainly slow down the reorientation dynamics of the hydroxyl group that points toward the anion. In both cases, motion along only one molecular axis is impeded, so that the hydration shell is best described as semirigid. In this semirigid hydration picture, water molecules in the first hydration shell show anisotropic reorientation, whereas water molecules outside the first hydration shell remain unaffected. The inferred anisotropy in molecular motion explains why terahertz dielectric relaxation spectroscopy, which probes dipolar relaxation, is more sensitive to cation hydration effects while femtosecond infrared pump-probe spectroscopy, which is sensitive to reorientation of hydroxyl groups, is more sensitive to anion hydration effects. We also show that dissolution of CsI-a salt for which both cation and anion are weakly hydrated-has little effect on water reorientation dynamics, with hydration water displaying dynamics that are similar to those in bulk water.

Serotonin transporter knockout and repeated social defeat stress: impact on neuronal morphology and plasticity in limbic brain areas.

Low expression of the human serotonin transporter (5-HTT) gene presumably interacts with stressful life events enhancing susceptibility for affective disorders. 5-Htt knockout (KO) mice display an anxious phenotype, and behavioural differences compared to wild-type (WT) mice are exacerbated after repeated loser experience in a resident-intruder stress paradigm. To assess whether genotype-dependent and stress-induced behavioural differences are reflected in alterations of neuronal morphology in limbic areas, we studied dendritic length and complexity of pyramidal neurons in the anterior cingulate and infralimbic cortices (CG, IL), hippocampus CA1 region, and of pyramidal neurons and interneurons in the lateral (La) and basolateral (BL) amygdaloid nuclei in Golgi-Cox-stained brains of male WT and 5-Htt KO control and loser mice. Spine density was analysed for IL apical and amygdaloid apical and basal pyramidal neuron dendrites. While group differences were absent for parameters analysed in CG, CA1 and amygdaloid interneurons, pyramidal neurons in the IL displayed tendencies to shorter and less spinous distal apical dendrites in 5-Htt KO controls, and to extended proximal dendrites in WT losers compared to WT controls. In contrast, spine density of several dendritic compartments of amygdaloid pyramids was significantly higher in 5-Htt KO mice compared to WT controls. While a tendency to increased spine density was observed in the same dendritic compartments in WT after stress, changes were lacking in stressed compared to control 5-Htt KO mice. Our findings indicate that disturbed 5-HT homeostasis results in alterations of limbic neuronal morphology, especially in higher spinogenesis in amygdaloid pyramidal neurons. Social stress leads to similar but less pronounced changes in the WT, and neuroplasticity upon stress is reduced in 5-Htt KO mice.

Cooperativity in ion hydration.

Despite prolonged scientific efforts to unravel the effects of ions on the structure and dynamics of water, many open questions remain, in particular concerning the spatial extent of this effect (i.e., the number of water molecules affected) and the origin of ion-specific effects. A combined terahertz and femtosecond infrared spectroscopic study of water dynamics around different ions (specifically magnesium, lithium, sodium, and cesium cations, as well as sulfate, chloride, iodide, and perchlorate anions) reveals that the effect of ions and counterions on water can be strongly interdependent and nonadditive, and in certain cases extends well beyond the first solvation shell of water molecules directly surrounding the ion.