Search Results
Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies
The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
Application of scanning electrochemical microscopy for topography imaging of supported lipid bilayers
Oxidative stress in cellular environments may cause lipid oxidation and membrane degradation. Therefore, studying the degree of lipid membrane morphological changes by reactive oxygen and nitrogen species will be informative in oxidative stress-based therapies. This study introduces the possibility of using scanning electrochemical microscopy as a powerful imaging technique to follow the topographical changes of a solid-supported lipid bilayer model induced by reactive species produced from gas plasma. The introduced strategy is not limited to investigating the effect of reactive species on the lipid bilayer but could be extended to understand the morphological changes of the lipid bilayer due to the action of membrane proteins or antimicrobial peptides.
Static Disorder in Excitation Energies of the Fenna-Matthews-Olson Protein: Structure-Based Theory Meets Experiment
Inhomogeneous broadening of optical lines of the Fenna-Matthews-Olson (FMO) light-harvesting protein is investigated by combining a Monte Carlo sampling of low-energy conformational substates of the protein with a quantum chemical/electrostatic calculation of local transition energies (site energies) of the pigments. The good agreement between the optical spectra calculated for the inhomogeneous ensemble and the experimental data demonstrates that electrostatics is the dominant contributor to static disorder in site energies. Rotamers of polar amino acid side chains are found to cause bimodal distribution functions of site energy shifts, which can be probed by hole burning and single-molecule spectroscopy. When summing over the large number of contributions, the resulting distribution functions of the site energies become Gaussians, and the correlations in site energy fluctuations at different sites practically average to zero. These results demonstrate that static disorder in the FMO protein is in the realm of the central limit theorem of statistics. © 2020 American Chemical Society.
Polarization-resolved second-harmonic generation imaging through a multimode fiber
Multimode fiber-based endoscopes have recently emerged as a tool for minimally invasive endoscopy in tissue, at depths well beyond the reach of multiphoton imaging. Here, we demonstrate label-free second-harmonic generation (SHG) microscopy through such a fiber endoscope. We simultaneously fully control the excitation polarization state and the spatial distribution of the light at the fiber tip, and we use this to implement polarization-resolved SHG imaging, which allows imaging and identification of structural proteins such as collagen and myosin. We image mouse tail tendon and heart tissue, employing the endoscope at depths up to 1 mm, demonstrating that we can differentiate these structural proteins. This method has the potential for enabling instant and in situ diagnosis of tumors and fibrotic conditions in sensitive tissue with minimal damage.
Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering
The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
Water Dynamics in the Hydration Shells of Biomolecules
The structure and function of biomolecules are strongly influenced by their hydration shells. Structural fluctuations and molecular excitations of hydrating water molecules cover a broad range in space and time, from individual water molecules to larger pools and from femtosecond to microsecond time scales. Recent progress in theory and molecular dynamics simulations as well as in ultrafast vibrational spectroscopy has led to new and detailed insight into fluctuations of water structure, elementary water motions, electric fields at hydrated biointerfaces, and processes of vibrational relaxation and energy dissipation. Here, we review recent advances in both theory and experiment, focusing on hydrated DNA, proteins, and phospholipids, and compare dynamics in the hydration shells to bulk water.
Amyloids: From molecular structure to mechanical properties
Many proteins of diverse sequence, structure and function self-assemble into morphologically similar fibrillar aggregates known as amyloids. Amyloids are remarkable polymers in several respects. First of all, amyloids can be formed from proteins with very different amino acid sequences; the common denominator is that the individual proteins constituting the amyloid fold predominantly into a β-sheet structure. Secondly, the formation of the fibril occurs through non-covalent interactions between primarily the β-sheets, causing the monomers to stack into fibrils. The fibrils are remarkably robust, considering that the monomers are bound non-covalently. Finally, a common characteristic of fibrils is their unbranched, straight, fiber-like structure arising from the intertwining of the multiple β-sheet filaments. These remarkably ordered and stable nanofibrils can be useful as building blocks for protein-based functional materials, but they are also implicated in severe neurodegenerative diseases. The overall aim of this article is to highlight recent efforts aimed at obtaining insights into amyloid proteins on different length scales. Starting from molecular information on amyloids, single fibril properties and mechanical properties of networks of fibrils are described. Specifically, we focus on the self-assembly of amyloid protein fibrils composed of peptides and denatured model proteins, as well as the influence of inhibitors of fibril formation. Additionally, we will demonstrate how the application of recently developed vibrational spectroscopic techniques has emerged as a powerful approach to gain spatially resolved information on the structure-function relation of amyloids. While spectroscopy provides information on local molecular conformations and protein secondary structure, information on the single fibril level has been developed by diverse microscopic techniques. The approaches to reveal basic mechanical properties of single fibrils like bending rigidity, shear modulus, ultimate tensile strength and fracture behavior are illustrated. Lastly, mechanics of networks of amyloid fibrils, typically forming viscoelastic gels are outlined, with a focus on (micro-) rheological properties. The resulting fundamental insights are essential for the rational design of novel edible and biodegradable protein-based polymers, but also to devise therapeutic strategies to combat amyloid assembly and accumulation during pathogenic disorders.
Polymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings
Implantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.
Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma
In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
Membrane technologies for lactic acid separation from fermentation broths derived from renewable resources
Lactic acid (LA) was produced on a pilot scale using a defined medium with glucose, acid whey, sugar bread and crust bread. The fermentation broths were then subjected to micro-and nanofiltration. Microfiltration efficiently separated the microbial cells. The highest average permeate flow flux was achieved for the defined medium (263.3 L/m2/h) and the lowest for the crust bread-based medium (103.8 L/m2/h). No LA losses were observed during microfiltration of the acid whey, whilst the highest retention of LA was 21.5% for crust bread. Nanofiltration led to high rejections of residual sugars, proteins and ions (sulphate, magnesium, calcium), with a low retention of LA. Unconverted sugar rejections were 100% and 63% for crust bread and sugar bread media respectively, with corresponding LA losses of 22.4% and 2.5%. The membrane retained more than 50% of the ions and proteins present in all media and more than 60% of phosphorus. The average flux was highly affected by the nature of the medium as well as by the final concentration of LA and sugars. The results of this study indicate that micro-and nanofiltration could be industrially employed as primary separation steps for the biotechnologically produced LA.