Atomic force microscopy (AFM) was used to study native cellulose films prepared from a bacterial cellulose source, Acetobacter xylinum, using a novel application of the Langmuir-Blodgett technique. These films allowed high-resolution AFM images of single fibers and their microfibril structure to be obtained. Two types of experiments were performed. First, the fibers were characterized using samples that were dried after LB deposition. Next, novel protocols that allowed us to image single fibers of cellulose in films that were never dried were developed. This procedure allowed us to perform in situ AFM imaging studies of the enzymatic hydrolysis of single cellulose fibers in solution using cellulolytic enzymes. The in situ degradation of cellulose fibers was monitored over a 9 h period using AFM. These studies provided the first direct, real-time images of the enzymatic degradation of a single cellulose fiber. We have demonstrated the tremendous potential of AFM to study the mechanism of the enzymatic digestion of cellulose and to identify the most effective enzymes for the digestion of various cellulose structures or isomorphs.
The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.
This work uses electrochemical surface sensitive vibrational spectroscopy to characterize the adsorption of a known metal nanoparticle stabilizer and growth director, 4-methoxypyridine (MOP). Surface enhanced infrared absorption spectroscopy (SEIRAS) is employed to study the adsorption of 4-methoxypyridine on gold films. Experiments are performed under electrochemical control and in different electrolyte acidities to identify both the extent of protonation of the adsorbed species as well as its orientation with respect to the electrode surface. No evidence of adsorbed conjugated acid is found even when the electrolyte pH is considerably lower than the pKa. Through an analysis of the transition dipole moments, determined from DFT calculations, the SEIRA spectra support an adsorption configuration through the ring nitrogen which is particularly dominant in neutral pH conditions. Adsorption is dependent on both the electrical state of the Au film electrode as well as the presence of ions in the electrolyte that compete for adsorption sites at positive potentials. Combined differential capacitance measurements and spectroscopic data demonstrate that both a horizontal adsorption geometry and a vertical adsorption phase can be induced, with the former being found on negatively charged surfaces in acidic media and the latter over a wide range of polarizations in neutral solutions.
Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir-Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.
Surface-enhanced infrared adsorption spectroscopy (SEIRAS) and neutron reflectometry (NR) were employed to characterize ubiquinone (UQ) containing hybrid bilayer membranes. The biomimetic membrane was prepared by fusing phospholipid vesicles on a hydrophobic octadecanethiol monolayer self-assembled on a thin gold film. Using SEIRAS, the assembly of the membrane is monitored in situ. The presence of ubiquinone is verified by the characteristic carbonyl peaks from the quinone ester. A well-ordered distal lipid leaflet results from fusion of vesicles with and without the addition of ubiquinone. With applied potential, the hybrid bilayer membrane in the absence of UQ behaves in the same way as previously reported solid supported phospholipid membranes. When ubiquinone is incorporated in the membrane, electric field induced changes in the distal leaflet are suppressed. Changes in the infrared vibrations of the ubiquinone due to applied potential indicate the head groups are located in both polar and nonpolar environments. The spectroscopic data reveal that the isoprenoid unit of the ubiquinone is likely lying in the midplane of the lipid bilayer while the head has some freedom to move within the hydrophobic core. The SEIRAS experiments show redox behavior of UQ incorporated in a model lipid membrane that are otherwise inaccessible with traditional electrochemistry techniques.
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