SynDLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features.

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial dynamin-like proteins are still poorly investigated. SynDLP, the dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. In addition to typical GD-GD contacts, such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids independent of nucleotides. The structural characteristics of SynDLP oligomers suggest it to be the closest known bacterial ancestor of eukaryotic dynamin.

Surface chemistry of the ladybird beetle adhesive foot fluid across various substrates.

Nature has coevolved highly adaptive and reliable bioadhesives across a multitude of animal species. Much attention has been paid in recent years to selectively mimic these adhesives for the improvement of a variety of technologies. However, very few of the chemical mechanisms that drive these natural adhesives are well understood. Many insects combine hairy feet with a secreted adhesive fluid, allowing for adhesion to considerably rough and slippery surfaces. Insect adhesive fluids have evolved highly specific compositions which are consistent across most surfaces and optimize both foot adhesion and release in natural environments. For example, beetles are thought to have adhesive fluids made up of a complex molecular mixture containing both hydrophobic and hydrophilic parts. We hypothesize that this causes the adhesive interface to be dynamic, with molecules in the fluid selectively organizing and ordering at surfaces with complimentary hydrophobicity to maximize adhesion. In this study, we examine the adhesive fluid of a seven-spotted ladybird beetle with a surface-sensitive analytical technique, sum frequency generation spectroscopy, as the fluid interacts with three substrates of varied wettabilities. The resulting spectra present no evidence of unique molecular environments between hydrophilic and hydrophobic surfaces but exhibit significant differences in the ordering of hydrocarbons. This change in surface interactions across different substrates correlates well with traction forces measured from beetles interacting with substrates of increasing hydrophobicities. We conclude that insect adhesion is dependent upon a dynamic molecular-interfacial response to an environmental surface.

Surface chemistry of the frog sticky-tongue mechanism.

Frogs capture their prey with a highly specialized tongue. Recent studies indicate this tongue is covered with fibril-forming mucus that acts as a pressure sensitive adhesive. However, no analysis of the interfacial chemistry of frog tongue mucus has been performed. The goal of this study is to examine the chemical structure of the surface of mucus after a tongue strike. Previous studies of mucus from other animals suggest that mucus from a frog's tongue consists of mucins-serine-, threonine-, and proline-rich glycoproteins. Therefore, the authors expect to observe chemical bonds associated with glycoproteins, as well as fibrils formed at the mucus-tongue interface. To test this hypothesis, they collected both near-edge x-ray absorption fine structure (NEXAFS) microscopy images and sum frequency generation (SFG) vibrational spectra from layers of mucus left after frog tongue strikes on cleaned glass slides. NEXAFS imaging demonstrates a uniform distribution of amide, hydroxyl, and carbon-carbon bonds across the mucus surface. Difference spectra of individual N1s and C1s K-edge spectra pulled from these images indicate a structure consistent with fibril formation as well as disorder of oligosaccharide groups near the mucus surface. C-H region SFG spectra reveal surface active modes which likely stem from serine and threonine within the mucin protein. Combined, this work suggests that glycoproteins are well-ordered at the mucus-tongue interface.

Repelling and ordering: the influence of poly(ethylene glycol) on protein adsorption.

Development of new materials for drug delivery and biosensing requires the fine-tuning of interfacial properties. We report here the influence of the poly(ethylene glycol) (PEG) grafting density in model phospholipid monolayers on the adsorption behavior of bovine serum albumin and human fibrinogen, not only with respect to the amount of adsorbed protein, but also its orientational ordering on the surface. As expected, with increasing interfacial PEG density, the amount of adsorbed protein decreases up to the point where complete protein repellency is reached. However, at intermediate concentrations, the net orientation of adsorbed fibrinogen is highest. The different proteins respond differently to PEG, not only in the amount of protein adsorbed, but also in the manner that proteins adsorb. The results show that for specific cases, tuning the interfacial PEG concentration allows to guide the protein adsorption configuration, a feature sought after in materials for both biosensing and biomedical applications.

A trough for improved SFG spectroscopy of lipid monolayers.

Lipid monolayers are indispensable model systems for biological membranes. The main advantage over bilayer model systems is that the surface pressure within the layer can be directly and reliably controlled. The sensitive interplay between surface pressure and temperature determines the molecular order within a model membrane and consequently determines the membrane phase behavior. The lipid phase is of crucial importance for a range of membrane functions such as protein interactions and membrane permeability. A very reliable method to probe the structure of lipid monolayers is sum frequency generation (SFG) vibrational spectroscopy. Not only is SFG extremely surface sensitive but it can also directly access critical parameters such as lipid order and orientation, and it can provide valuable information about protein interactions along with interfacial hydration. However, recent studies have shown that temperature gradients caused by high power laser beams perturb the lipid layers and potentially obscure the spectroscopic results. Here we demonstrate how the local heating problem can be effectively reduced by spatially distributing the laser pulses on the sample surface using a translating Langmuir trough for SFG experiments at lipid monolayers. The efficiency of the trough is illustrated by the detection of enhanced molecular order due to reduced heat load.

Nitrated Fatty Acids Modulate the Physical Properties of Model Membranes and the Structure of Transmembrane Proteins.

Nitrated fatty acids (NO2 -FAs) act as anti-inflammatory signal mediators, albeit the molecular mechanisms behind NO2 -FAs' influence on diverse metabolic and signaling pathways in inflamed tissues are essentially elusive. Here, we combine fluorescence measurements with surface-specific sum frequency generation vibrational spectroscopy and coarse-grained computer simulations to demonstrate that NO2 -FAs alter lipid organization by accumulation at the membrane-water interface. As the function of membrane proteins strongly depends on both, protein structure as well as membrane properties, we consecutively follow the structural dynamics of an integral membrane protein in presence of NO2 -FAs. Based on our results, we suggest a molecular mechanism of the NO2 -FA in vivo activity: Driven by the NO2 -FA-induced lipid layer reorganization, the structure and function of membrane-associated (signaling) proteins is indirectly affected.

Identifying the selectivity of antimicrobial peptides to cell membranes by sum frequency generation spectroscopy.

Cationic amphiphilic peptides have been engineered to target both Gram-positive and Gram-negative bacteria while avoiding damage to other cell types. However, the exact mechanism of how these peptides target, bind, and disrupt bacterial cell membranes is not understood. One specific peptide that has been engineered to selectively capture bacteria is WLBU2 (sequence: RRWVRRVRRWVRRVVRVVRRWVRR). It has been suggested that WLBU2 activity stems from the fact that when interacting with bacterial cell membranes the peptide assumes an α-helical structure and inserts itself into the membrane. Alternatively, in the presence of mammalian cell membranes, the peptide assumes an inert ß-sheet structure. To test this hypothesis, the authors applied sum frequency generation (SFG) spectroscopy and surface tensiometry to identify the structure of WLBU2 as it interacts with model lipid monolayers that mimic mammalian and bacterial cell membranes. Model mammalian cell membranes were built upon zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids while bacterial cell membranes were constructed with negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) lipids. Observed changes in surface pressure at the peptide-lipid-air interface demonstrate that the peptide has a clear binding preference toward negatively charged bacteria-like lipids. The structure of both the lipids and peptides were characterized by SFG spectra collected at the monolayer interface. Changes in monolayer structure as the peptide binds were observed by tracking the intensities of SFG vibrational modes related to the acyl chains within the lipids. Peptide structures when bound to both types of lipids were determined by SFG spectra collected within the amide I vibrational band. The SFG spectra of WLBU2 interacting with the model mammalian lipid monolayer contain two peaks near 1642 and 1678 cm-1 indicative of an inactive ß-sheet structure. SFG spectra collected from the peptide bound to a bacteria-like lipid monolayer contains just a single peak near 1651 cm-1 which corresponds to an active α-helix structure. Combined, the tensiometry and SFG results demonstrate that WLBU2 both possesses a higher binding affinity toward and is in an active α-helix structure when bound to bacterial cell membranes.

SAP(E) - A cell-penetrating polyproline helix at lipid interfaces.

Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides - SAP(E) - which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly - since peptide-membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur.

Oppositely Charged Ions at Water-Air and Water-Oil Interfaces: Contrasting the Molecular Picture with Thermodynamics.

The surface-active ions tetraphenylarsonium (Ph4As(+)) and tetraphenylboron (Ph4B(-)) have a similar structure but opposite charge. At the solution-air interface, the two ions affect the surface tension in an identical manner, yet sum-frequency generation (SFG) spectra reveal an enhanced surface propensity for Ph4As(+) compared with Ph4B(-), in addition to opposite alignment of interfacial water molecules. At the water-oil interface, the interfacial tension is 7 mN/m lower for Ph4As(+) than for Ph4B(-) salts, but this can be fully accounted for by the different bulk solubility of these ions in the hydrophobic phase, rather than inherently different surface activities. The different solubility can be accounted for by differences in electronic structure, as evidenced by quantum chemical calculations and NMR studies. Our results show that the surface propensity concluded from SFG spectroscopy does not necessarily correlate with interfacial adsorption concluded from thermodynamic measurements.

Reversible activation of a cell-penetrating peptide in a membrane environment.

Cell-penetrating peptides (CPPs) are promising molecules as drug carriers. However, because their uptake mainly involves endocytic mechanisms, endosomal trapping of the carrier (and drug) remains a high barrier for biomedical applications. The viral fusion mimic GALA, a pH-triggered CPP, takes advantage of the decreasing pH during endosome maturation to selectively attack endosomal membranes. Below pH 6, the sequence folds into a helix and can disrupt membranes. In this study, we show that the lipid bilayer radius-of-curvature has a negligible effect on GALA-induced leakage kinetics and that GALA remains pH responsive after inserting into a lipid membrane. The peptide can be reversibly "switched" between its inactive and active states after incorporation into the hydrophobic environment of lipid membranes, even after substantially interacting with lipid chains. This ability makes GALA-based delivery a potentially safe and efficient strategy for endosomal escape.

No photocatalyst required--versatile, visible light mediated transformations with polyhalomethanes.

A visible light mediated, but photocatalyst-free method for the oxidative α-CH functionalization of tertiary amines with a broad scope of carbon- and heteroatom nucleophiles using polyhalomethanes has been developed. In addition, the pivotal visible light triggered activation of polyhalomethanes offers mild conditions for efficient Kharasch-type additions onto non-activated olefins. Preliminary mechanistic studies are reported.

Full membrane spanning self-assembled monolayers as model systems for UHV-based studies of cell-penetrating peptides.

Biophysical studies of the interaction of peptides with model membranes provide a simple yet effective approach to understand the transport of peptides and peptide based drug carriers across the cell membrane. Herein, the authors discuss the use of self-assembled monolayers fabricated from the full membrane-spanning thiol (FMST) 3-((14-((4'-((5-methyl-1-phenyl-35-(phytanyl)oxy-6,9,12,15,18,21,24,27,30,33,37-undecaoxa-2,3-dithiahenpentacontan-51-yl)oxy)-[1,1'-biphenyl]-4-yl)oxy)tetradecyl)oxy)-2-(phytanyl)oxy glycerol for ultrahigh vacuum (UHV) based experiments. UHV-based methods such as electron spectroscopy and mass spectrometry can provide important information about how peptides bind and interact with membranes, especially with the hydrophobic core of a lipid bilayer. Near-edge x-ray absorption fine structure spectra and x-ray photoelectron spectroscopy (XPS) data showed that FMST forms UHV-stable and ordered films on gold. XPS and time of flight secondary ion mass spectrometry depth profiles indicated that a proline-rich amphipathic cell-penetrating peptide, known as sweet arrow peptide is located at the outer perimeter of the model membrane.