Single Molecule Measurements of the Accessibility of Molecular Surfaces

An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method (“Q-SLIP”, Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.

Physicochemical Characteristics of Fungal Xylanases and their Potential for Biobleaching of Kraft and Non-wood Pulps

Xylanases are enzymes with a wide variety of biotechnological applications, such as in the bioconversion of lignocellulosic materials, improvement of feed digestibility, and bleaching kraft pulps to increase pulp brightness. Many studies have been conducted and published over the years on cellulose pulp bleaching due to the need to search for more sustainable tools and thus reduce environmental pollution. Thus, in this review, we focus on analyzing the biochemical properties of xylanases produced by mesophilic and thermophilic fungi that have been used in the bleaching processes of kraft and non-wood pulps. Eucalyptus kraft pulp is still one of the most used raw materials in the production of pulp and paper, while straw and bagasse are alternative sources of non-wood pulps. Thermophilic fungal xylanases show optimum enzymatic activity at high temperatures and a shorter treatment period when compared to mesophilic xylanases in the bleaching step. However, mesophilic fungal xylanases exhibit a longer period of enzymatic treatment but achieve a satisfactory brightness and kappa number. Hence, these approaches will contribute to future applications of these xylanolytic enzymes in pulp and paper industries.

High-Resolution Peat Core Chronology Covering the Last 12 KYR Applying an Improved Peat Bog Sampling

ABSTRACTThis work focuses on building a high-resolution age-depth model for quantitative palaeoclimate study from the Mohos peat bog, East Carpathian mountains. The investigated core presents a continuous peat profile for the last 12 kyr. The chronology was based on 36 accelerator mass spectrometry radiocarbon (AMS 14C) analyses of the separated Sphagnum samples from different depths of the profile. Dry Sphagnum samples for AMS dating were prepared using the classic acid-base-acid (ABA) method followed with an oxidative bleaching step to get clean cellulose. Sphagnum cellulose samples were measured by AMS using the EnvironMICADAS at the ICER (Debrecen, Hungary). A high-resolution chronology was obtained with the use of Bayesian age-depth modeling. Peat accumulation rate has been calculated and the sections with variable accumulation rate values were observed along the profile.

Effects of cultivation, washing, and bleaching conditions on bacterial cellulose fabric production

This study aims to compare different conditions in the three-step (cultivation, washing, and bleaching) production of white bacterial cellulose (BC) fabric to introduce it as a new type of fabric in the textile industry. The BC fabric was evaluated on the basis of its surface morphology and chemical structure. The “production BC” after the cultivation step was cultured using glucose as the carbon source in the Hestrin–Schramm (HS) medium. It was produced with the highest production yield (33.2 ± 6.85%), the highest thickness (0.35 ± 0.09 mm), and the flattest surface (211 nm). The bacteria remaining on “washed BC” after the washing step were washed out using 3% NaOH solution, and the nanoscale network structure maintained its integrity after washing. The white BC fabric after the bleaching step was bleached using 5% H2O2 solution. The white BC fabric with the highest white index (73.15 ± 1.09%) without a natural yellowish-brown color was produced. In the Fourier transform infrared spectroscopy (FTIR) spectra of the white BC fabric, the peaks of proteins and amino acids derived from the bacteria disappeared, while the cellulose I crystal structure was maintained. Also, X-ray diffraction analysis showed that the crystallinity of the white BC fabric increased compared to that of the control sample, and the highest crystallinity of 80.6% was obtained.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach

Correlating single-molecule fluorescence photo-bleaching step analysis and single-molecule super-resolution imaging, our findings for the clustering effect of the NMDA receptor ion channel on the live cell membranes provide a new and significant understanding of the structure–function relationship of NMDA receptors.