Protein Monolayer Research Articles

Encapsulation of cannabidiol in hemp seed oleosomes

Oleosomes are natural lipid droplets that can be extracted intact from oil seeds, forming oil/water emulsions. Their lipid cores, surrounded by a monolayer of phospholipids and proteins, make oleosomes suitable as carriers of hydrophobic bioactive compounds like cannabidiol (CBD). As CBD is crystalline at room temperature, it first has to be liquified to allow better encapsulation. This was done by heating (80 °C for 4 h) or by pre-solubilizing CBD in ethanol and then the liquified CBD was mixed with oleosome dispersions for the encapsulation. Both methods exhibit good encapsulation efficiency, but the results were significantly influenced by the ratio of CBD to lipid contents, regardless of the encapsulation method applied. At higher concentrations of CBD relative to that of the lipid in the oleosomes, the encapsulation efficiency decreased as saturation was attained. Moreover, the in vitro digestion analysis was conducted to investigate the potential of oleosomes as carriers to transport CBD. The relatively slow and steady release of CBD from oleosomes indicates that oleosomes are a slow-release carrier for hydrophobic functional ingredients. An important finding is that the encapsulation and in vitro digestive properties of the oleosomes remain unaffected by the presence of CBD, heating treatment or ethanol, which could bring more opportunities for the applications of oleosomes as carriers in various fields.

Fabrication of electrospun ion exchanger adsorbents with morphologies designed for the separation of proteins and plasmid DNA

Electrospun cellulose adsorbents are an emergent class of materials applied to a variety of bioprocess separations as an analogue to conventional packed bed chromatography. Electrospun adsorbents have proven to be effective as rapid cycling media, enabling high throughput separation of proteins and viral vectors without compromising selectivity and recovery. However, there is a current lack of knowledge in relation to the manipulation and control of electrospun adsorbent structure with function and performance to cater to the separation needs of emerging, diverse biological products.In this study, a series of electrospun cellulose adsorbents were fabricated by adjusting their manufacturing conditions. A range of fiber diameters (400 to 600 nm) was created by changing the electrospinning polymer solution. Additionally, a range of porosities (0.4 to 0.7 v/v) was achieved by varying the laminating pressures on the electrospun sheets. The adsorbents were functionalized with different degrees of quaternary amine ligand density to create 18 prototype anion exchangers. Their morphology was characterized by BET nitrogen adsorption surface area, X-ray computed tomography, capillary flow porometry and scanning electron microscopy measurements.The physical characteristics of the adsorbents were used in an adapted semi-empirical model and compared to measured permeability data. Permeabilities of prototypes ranged from 10−2 to 10−4 mDarcy. The measured data showed good adherence to modelled data with possible improvements in acquiring wet adsorbent characteristics instead of dried material. Finally, the electrospun adsorbents were characterized for their binding capacity of model proteins of different sizes (diameters of 3.5 nm and 8.9 nm) and plasmid DNA. Static binding capacities ranged from 5 mg/ml to 25 mg/ml for the proteins and plasmid DNA and showed <20 % deviation from monolayer coverage based on BET surface area. Therefore, it was concluded that the electrospun adsorbents most likely adsorb monolayers of proteins and plasmid DNA on the surface with minimal steric hindrance.

Investigation of numerical transport models in protein-based molecular junctions with cofactors of diverse chemical natures

The cofactors of proteins dictate the charge transport mechanism across molecular junctions when self-assembled protein monolayers are sandwiched between two metal electrodes. Here, we summarized how the chemical coordination nature of cofactors in various proteins modulates electrical conductance by investigating electronic transport studies across different protein-based molecular junctions under various forces applied under the AFM tip. We have utilized several numerical techniques of electronic transport to analyse the experimentally obtained current–voltage measurements across various protein-based molecular junctions and depicted the origin of electronic modulation in the electrical conductance under different external stimuli. We could also find the origin of electronic conductance modulation under external stimuli at various applied forces by obtaining several analytical transport parameters such as energy barrier, coupling strength, and electrical conductance values. Utilizing density-functional-theory calculations, we further validate that the electronic density of states present in the cofactors within the proteins dominates the electronic transport behaviours across protein-based molecular junctions. Our findings reveal the limiting factor for applying various external stimuli on different proteins, which could be further valuable in bioelectronic applications. We have also found that the organic cofactor containing protein follows all the tunneling mechanism-related numerical transport models and the electronic transport across proteins with pure inorganic cofactors follows Landauer transport formalism.

Dimeric and monomeric conformation of SARS-CoV-2 main protease: New technical approaches based on IR radiation

The main proteases Mpro are a group of highly conserved cysteine hydrolases in β-coronaviruses. They have been demonstrated to play an unavoidable role in viral replication, and consequently they have been suggested as key targets for treating coronavirus-caused infectious diseases, mainly from the COVID-19 epidemic. Since the most functional form for Mpro enzymatic activity is associated to its homodimer, compounds inhibiting dimerization should also inhibit catalytic activity. We show how PIR-SEIRA (Plasmonic Internal Reflection-Surface Enhanced InfraRed Absorption) spectroscopy can be a noteworthy technique to study proteins subtle structural variations associated to inhibitor binding. Nanoantennas arrays can selectively confine and enhance electromagnetic field via localized plasmonic resonances, thus promoting ultrasensitive detection of biomolecules in close proximity of nanoantenna arrays and enabling the effective investigation of protein monolayers. By adopting this approach, reflection measurements conducted under back illumination of nanoantennas allow to probe anchored protein monolayers, with minimum contribution of environmental buffer molecules. PIR-SEIRA spectroscopy on Mpro was carried out by ad hoc designed devices, resonating in the spectral region of Amide I and Amide II bands. We evaluated here the structure of anchored monomers and dimers in different buffered environment and in presence of a newly designed Mpro inhibitor. Experimental results show that dimerization is not associated to relevant backbone rearrangements of the protein at secondary structure level, and even if the compound inhibits the dimerization, it is not effective at breaking preformed dimers.

Protein Adsorption on Solid Surfaces: Data Mining, Database, Molecular Surface-Derived Properties, and Semiempirical Relationships.

Protein adsorption on solid surfaces is a process relevant to biological, medical, industrial, and environmental applications. Despite this wide interest and advancement in measurement techniques, the complexity of protein adsorption has frustrated its accurate prediction. To address this challenge, here, data regarding protein adsorption reported in the last four decades was collected, checked for completeness and correctness, organized, and archived in an upgraded, freely accessible Biomolecular Adsorption Database, which is equivalent to a large-scale, ad hoc, crowd-sourced multifactorial experiment. The shape and physicochemical properties of the proteins present in the database were quantified on their molecular surfaces using an in-house program (ProMS) operating as an add-on to the PyMol software. Machine learning-based analysis indicated that protein adsorption on hydrophobic and hydrophilic surfaces is modulated by different sets of operational, structural, and molecular surface-based physicochemical parameters. Separately, the adsorption data regarding four "benchmark" proteins, i.e., lysozyme, albumin, IgG, and fibrinogen, was processed by piecewise linear regression with the protein monolayer acting as breakpoint, using the linearization of the Langmuir isotherm formalism, resulting in semiempirical relationships predicting protein adsorption. These relationships, derived separately for hydrophilic and hydrophobic surfaces, described well the protein concentration on the surface as a function of the protein concentration in solution, adsorbing surface contact angle, ionic strength, pH, and temperature of the carrying fluid, and the difference between pH and the isoelectric point of the protein. When applying the semiempirical relationships derived for benchmark proteins to two other "test" proteins with known PDB structure, i.e., β-lactoglobulin and α-lactalbumin, the errors of this extrapolation were found to be in a linear relationship with the dissimilarity between the benchmark and the test proteins. The work presented here can be used for the estimation of operational parameters modulating protein adsorption for various applications such as diagnostic devices, pharmaceuticals, biomaterials, or the food industry.

Formation of a ‘protein corona’ on the Fe3O4@SiO2 nanoparticles surface

The patterns of interaction of the nanoparticles surface with protein components of biosystems have been studied on the example of the interaction of magnetic nanoparticles of the core–shell structure with albumin. Data on particle sizes and their spectra in the visible region in the presence of different quantity of protein have been obtained and analyzed by sorption, spectral and dynamic light scattering methods and albumin sorption isotherms in various media. The formation of the ordered protein monolayer (so-called ‘hard corona’) with subsequent forming of the multilayer of a different structure (‘soft corona’) has been experimentally shown; it demonstrates that it is possible to computationally distinguish between mono- and multi-layer using data from sorption experiments.

Morphology Observation of Two-Dimensional Monolayers of Model Proteins on Water Surface as Revealed by Dropping Method.

We have investigated the morphology of two-dimensional monolayers of gramicidin-D (GD) and alamethicin (Al) formed on the water surface by the dropping method (DM) using surface tension measurement (STm), Brewster angle microscopy (BAM), and atomic force microscopy (AFM). Dynamic light scattering (DLS) revealed that GD in alcoholic solutions formed a dimeric helical structure. According to the CD and NMR spectroscopies, GD molecules existed in dimer form in methanol and lipid membrane environments. The STm results and BAM images revealed that the GD dimer monolayer was in a liquid expanded (LE) state, whereas the Al monolayer was in a liquid condensed (LC) state. The limiting molecular area (A0) was 6.2 ± 0.5 nm2 for the GD-dimer and 3.6 ± 0.5 nm2 for the Al molecule. The AFM images also showed that the molecular long axes of both the GD-dimer and Al were horizontal to the water surface. The stability of each monolayer was confirmed by the time dependence of the surface pressure (π) observed using the STm method. The DM monolayer preparation method for GD-dimer and Al peptide molecules is a useful technique for revealing how the model biological membrane's components assemble in two dimensions on the water surface.

Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this “spectator protein”, exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Synthesized complex-frequency excitation for ultrasensitive molecular sensing

Sensors have emerged as indispensable analytical tools across a wide range of important fields, encompassing environmental monitoring, food safety, and public health. They facilitate early disease diagnosis, personalized medicine, and rapid detection of toxic agents. However, detecting trace molecules remains a significant challenge. Surface-enhanced infrared absorption (SEIRA) based on plasmonic nanostructures, particularly graphene, has emerged as a promising approach to enhance sensing sensitivity. While graphene-based SEIRA offers advantages such as high sensitivity and active tunability, intrinsic molecular damping weakens the interaction between vibrational modes and plasmons. Here, we demonstrate ultrahigh-sensitive molecular sensing based on synthesized complex-frequency waves (CFW). Our experiment shows that CFW can amplify the molecular signals (silk protein monolayer) detected by graphene-based sensor by at least an order of magnitude and can be universally applied to molecular sensing in different phases. Our approach is highly scalable and can facilitate the investigation of light-matter interactions, enabling diverse potential applications in fields such as optical spectroscopy, biomedicine and pharmaceutics.

Protein/Protein and Quantum Dot/Protein Organization in Sequential Monolayer Materials Studied Using Resonance Energy Transfer

Controlled junctions of proteins and nanomaterials offer multiple potential applications in the further construction of nanobiodevices. One of the possible junction types is a set of sequential monolayers of various components deposited on a given substrate. The advantage of such an organization is its high sensitivity, resulting from a huge surface covered by molecules or particles. What is more, the molecules/particles adsorbed on a substrate might be easier to handle than the assay in a cuvette. For further application, there should be crosstalk between monolayers; this is defined by the type of individuals forming a complex system. Here, we are studying, using mainly confocal microscopy and FLIM imaging, crosstalk through resonance energy transfer. The sequential monolayers of fluorescent proteins and CdTe quantum dots were deposited on a convenient substrate, a polyvinylidene difluoride membrane. First, we found that the degree of coverage is lower in the second monolayer. Hence, by manipulating the order of deposition, we obtained a system with a varied yield of resonance energy transfer with a donor excess or an acceptor excess. For a deeper understanding of the energy transfer and its limitations in this system influencing the assay pursuit, we utilized Monte Carlo computation. We found that, indeed, the distance between the monolayers, as well as the degree of coverage, is crucial. With the results of the simulation, we might estimate the relative degree of coverage in our sequential monolayers. We also found that in quantum-dots/protein-composed systems, the yield is stronger than predicted by Monte Carlo simulation. Hence, there should be protein reorientation on the nanoparticle surface, leading to such an effect. Finally, we showed that the yield of resonance energy transfer may be modulated by the external application of poly-L-lysines. These chemicals influenced QD fluorescence but not protein fluorescence and might be used, therefore, as a trigger or a switch in nanobiodevices employing those types of sequential monolayers.

Study on the interfacial properties of rice bran oil bodies affecting their physicochemical properties, stability and adsorption behavior at the oil-water interface for the design of precisely emulsified systems

Plant oil bodies (OBs) consist of a continuous monolayer of phospholipids-interfacial proteins covering the central triglycerides, which confers them extreme physicochemical stability. In this paper, rice bran OBs (RBOBs) extracted with NaHCO3 as a medium at pH 7.5–11.0 were naturally pre-emulsified spherical droplets with integral interfacial structures. The extraction pH determined the physicochemical properties, stability and interfacial behavior of RBOBs by affecting their interfacial properties. RBOBs extracted at pH 7.5 had more protein chains inserted into the acyl chains of the lipids, causing the RBOBs to be more stable, whereas at pH 11.0, the contents of PL and protein supported on the surface of TAGs core decreased significantly, the interfacial proteins were more flexible and looser, and part of the hydrophobic amino acids originally inserted in the TAGs core were exposed, destabilizing the OBs. RBOBs could spontaneously adsorb at the oil-water interface and form an interfacial film with elasticity as the primary feature. The structural rearrangement of the surface-active components was crucial to the formation of the interfacial membrane. More small-sized OBs like Pickering stabilizers adsorbed intact at the oil-water interface, creating ordered dense film (monolayer) particles. In contrast, large-sized RBOBs readily ruptured with a more pronounced degree of structural rearrangement of phospholipid-protein membrane fragments and disordered interfacial adsorption behavior. The results provide a reference for the development of precisely emulsified systems based on the special structure of plant OBs.

Bacterial extracellular electron transfer components are spin selective.

Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway.

A graphene-metal hybrid biosensor for SEIRA spectroscopy applications

Detecting the building blocks of all living organisms plays a vital role in medical diagnosis and biological research. Infrared (IR) spectroscopy is one of the techniques for detecting biomolecules, such as DNA, RNA, and lipids, through their vibrational fingerprints. IR spectroscopy provides biosensing by directly measuring changes in absorption spectra; however, nanometric-size molecules reveal a weak signal-to-noise ratio with IR light, which is a disadvantage for sensitive bio-detection. Surface-Enhanced Infrared Spectroscopy (SEIRA) based on different structures and materials has been widely used to overcome this problem. In this study, a SEIRA platform based on a hybrid graphene-metal nanoantenna array is designed by combining the enhanced sensitivity of graphene with its electrical tunability and strong-field enhancement with metallic structures. The plasmonic resonance of graphene nano-slit is dynamically tuned to detect fingerprints of protein monolayer at different frequencies. The same detection behavior has been statically demonstrated by metallic antennas. Our results indicate that the sensitivity of the SEIRA sensor platform is quite high due to the combination of graphene and metal hybrid structure compared to the metal resonator alone because of the strong light confinement in the graphene layer. The proposed biosensor can detect the Amid-I and Amid-II bonds, which create relatively strong vibrational fingerprints, as well as the Amide-III bond, which is a very weak bond of a monolayer protein-goat-mouse Immunoglobulin (IgG) protein. It can also be a very useful characterization tool for identifying the molecular fingerprints of unknown chemicals and molecules with its capability to span a large spectral range either dynamically or statically. The strong light absorption via metallic structures compensates for the large areal coverage of graphene sheets, which is quite a challenge for dynamic tuning in real applications.

Protein Adsorption on Carboxy-Functionalized Microparticles Revealed by Zeta Potential and Absorption Spectroscopy Measurements

Abstract Addition of micro/nanoparticles to a protein solution leads to the formation of a protein layer on the particle surface, called a protein corona. We investigate here the adsorption behavior of myoglobin, hemoglobin, cytochrome-c, and lysozyme on carboxy-functionalized polystyrene microparticles using zeta potential and absorption spectroscopy measurements. The observed adsorption behavior differs according to the method of detection: monolayer for zeta potential and multilayer for absorption spectroscopy. Langmuir-type monolayer adsorption is observed with zeta potential measurements, because zeta potential (ζ) responds only to the charge density at the outermost protein layer. Multilayer adsorption is observed by absorption spectroscopy. Spectroscopic results were analyzed by the Guggenheim-Anderson-de Boer (GAB) model, which comprises a hard corona formed by strong interaction between the protein and the particle surface and a soft corona formed by weak interaction between adsorbed and bulk-solution proteins. The extent of hard and soft corona formation depends on pH. When a protein monolayer is prepared by covalent modification of the particle surface, the number of layers in the protein corona decreases relative to the case of protein adsorption on bare particles. This result demonstrates that electrostatic interactions between the protein and particle surface play a key role in the formation of a protein corona.

Simultaneous detection of molecules with the surface-enhanced infrared absorption sensor platform based on disk antennas with double spacer

Biomolecule detection has become important in many applications such as medical diagnosis, forensic analysis, basic biological studies, and food quality assessment. In particular, the Mid-infrared range offers an important opportunity for biomolecular sensing as it covers the molecular vibrational spectra of vital biochemicals such as Deoxyribonucleic acid, Ribonucleic Acid, and proteins. In this study, a double band absorbing plasmonic nanoantenna array with two gold disk resonators is proposed. The biosensing ability of this structure was investigated using the protein-goat anti-mouse immunoglobulin G model and Polymethyl methacrylate film. The basic structural bonds of protein monolayer, namely Amide-I, Amide-II, and Amide-III showed vibrational signatures at 6010 nm (∼1664 cm−1), 6496 nm (∼1539 cm−1), and 6989 nm (∼1431 cm−1) wavelengths, respectively. In addition, the spectral response of the proposed antenna structure was investigated using a Polymethyl methacrylate film by detecting the C=O and the C-H bonds. The strong dipole moment at C=O showed a strong absorption deep at 5782 nm (∼1730 cm−1) while the C-H bond has shown a relatively low absorption deep at 3350 nm (∼2985 cm−1) and 3395 nm (∼2946 cm−1). Our findings indicate that the double spacer disk configuration detects the spectral signature of the protein monolayer and Polymethyl methacrylate film in each band, simultaneously. The dual-band can be tuned independently by carefully engineering the radii of the double disks without making an effect on the other band. The proposed structure can be used as a characterization tool for identifying unknown complex molecules by simply detecting their spectral fingerprints in each mode of the dual-band, independently. Also, this design strategy can be insight to multi-mode SEIRA platforms, where more complex chemical molecules are needed to be detected or identified in biology, chemistry, and defense areas.

Cholesterol esters lipid droplets biogenesis.

Lipid droplets (LDs) are nearly universal organelles, present in most of the eukaryotic cells. As storage organelles, they are the cornerstone of lipid storage and homeostasis. They consist of a neutral lipid core surrounded by a phospholipid and protein monolayer. Triacylglycerols (TGs) and Cholesteryl Esters (CEs) are the two most abundant neutral lipids. While TG melts at 4°C, CE melts at 44°C, raising the question of how cells manage to package CE in micron-sized LDs. In order to understand the biogenesis of CE-rich LDs, we first took advantage of in-vitro systems, such as Giant Unilamellar Vesicles (GUVs) and Droplet Embedded Vesicles (DEVs), to better study and predict the behaviour of CE molecules confined in a membrane bilayer. Then, we conducted cell experiments which agreed with our in-vitro models. Our studies revealed that CEs form supercooled droplets when representing more than 20% of the LDs’ lipids and, in particular, liquid-crystalline phases if above 90%. In model bilayers, CE condensed and nucleated as droplets when a critical CE/Phospholipids ratio was reached. We also found that this threshold was reduced in the presence of TG. Accordingly, CE LDs nucleation was strongly reduced upon inhibition of TG synthesis in cells. Our data point to a unique model whereby TG catalyzes the nucleation of CE LDs.

Tripeptide Self-Assembled Monolayers as Biocompatible Surfaces for Cytochrome c Electrochemistry.

Biocompatible tripeptide self-assembled monolayers (SAMs) are designed with a carboxylate group on the terminal amino acid (glutamate, aspartate, or amino adipate) to electrostatically attract the lysine groups around the heme crevice in horse heart cytochrome c (cyt c), creating an electroactive protein/tripeptide/Au interfacial structure. Exposing the peptide/Au electrode to cyt c resulted in an 11 ± 3 pmol/cm2 electroactive protein surface coverage. Topographical images of the interfacial structure are obtained down to single-protein resolution by atomic force microscopy. Uniform protein monolayer assemblies are formed on the Au electrode with no major surface roughness changes. The cyt c/peptide/Au electrode systems were examined electrochemically to probe surface charge effects on the redox thermodynamics and kinetics of cyt c. Neutralization of protein surface charge due to adsorption on anionic COOH-terminated SAMs was found to change the formal potential, as determined by cyclic voltammetry. The cyt c/peptide/Au electrodes exhibit formal potentials shifted to more positive values, have a surface carboxylic acid pKa of 6 or higher, and produce effective cyt c surface charges (Zox) of -6 to -14. The Marcus theory is utilized to determine the protein electron transfer rates, which are ∼5 times faster for cyt c/tripeptide/Au compared to cyt c/11-mercaptoundecanoic acid SAMs of similar chain lengths.

Biotin Binding Hardly Affects Electron Transport Efficiency across Streptavidin Solid-State Junctions

The electron transport (ETp) efficiency of solid-state protein-mediated junctions is highly influenced by the presence of electron-rich organic cofactors or transition metal ions. Hence, we chose to investigate an interesting cofactor-free non-redox protein, streptavidin (STV), which has unmatched strong binding affinity for an organic small-molecule ligand, biotin, which lacks any electron-rich features. We describe for the first time meso-scale ETp via electrical junctions of STV monolayers and focus on the question of whether the rate of ETp across both native and thiolated STV monolayers is influenced by ligand binding, a process that we show to cause some structural conformation changes in the STV monolayers. Au nanowire-electrode-protein monolayer-microelectrode junctions, fabricated by modifying an earlier procedure to improve the yields of usable junctions, were employed for ETp measurements. Our results on compactly integrated, dense, uniform, ∼3 nm thick STV monolayers indicate that, notwithstanding the slight structural changes in the STV monolayers upon biotin binding, there is no statistically significant conductance change between the free STV and that bound to biotin. The ETp temperature (T) dependence over the 80-300 K range is very small but with an unusual, slightly negative (metallic-like) dependence toward room temperature. Such dependence can be accounted for by the reversible structural shrinkage of the STV at temperatures below 160 K.

Phospholipids diffusion on the surface of model lipid droplets

Lipid droplets (LD) are organelles localized in the membrane of the Endoplasmic Reticulum (ER) that play an important role in metabolic functions. They consist of a core of neutral lipids surrounded by a monolayer of phosphoplipids and proteins resembling an oil-in-water emulsion droplet. Many studies have focused on the biophysical properties of these LDs. However, despite numerous efforts, we are lacking information on the mobility of phospholipids on the LDs surface, although they may play a key role in the protein distribution. In this article, we developed a microfluidic setup that allows the formation of a triolein–buffer interface decorated with a phospholipid monolayer. Using this setup, we measured the motility of phospholipid molecules by performing Fluorescent Recovery After Photobleaching (FRAP) experiments for different lipidic compositions. The results of the FRAP measurements reveal that the motility of phospholipids is controlled by the monolayer packing decorating the interface.

The Bacillus anthracis S-layer is an exoskeleton-like structure that imparts mechanical and osmotic stabilization to the cell wall.

Surface layers (S-layers) are 2D paracrystalline protein monolayers covering the cell envelope of many prokaryotes and archaea. Proposed functions include a role in cell support, as scaffolding structure, as molecular sieve, or as virulence factor. Bacillus anthracis holds two S-layers, composed of Sap or EA1, which interchange in early and late exponential growth phase. We previously found that acute disruption of B. anthracis Sap S-layer integrity, by means of nanobodies, results in severe morphological cell surface defects and cell collapse. Remarkably, this loss of function is due to the destruction of the Sap lattice structure rather than detachment of monomers from the cell surface. Here, we combine force nanoscopy and light microscopy observations to probe the contribution of the S-layer to the mechanical, structural, and functional properties of the cell envelope, which have been so far elusive. Our experiments reveal that cells with a compromised S-layer lattice show a decreased compressive stiffness and elastic modulus. Furthermore, we find that S-layer integrity is required to resist cell turgor under hypotonic conditions. These results present compelling experimental evidence indicating that the S-layers can serve as prokaryotic exoskeletons that support the cell wall in conferring rigidity and mechanical stability to bacterial cells.