Results for “Protein adsorption” — Page 4

DOE OSTI2023

Inhibition of Extracellular Enzyme Activity by Reactive Oxygen Species upon Oxygenation of Reduced Iron-Bearing Minerals

The dual roles of minerals in inhibiting and prolonging extracellular enzyme activity in soils and sediments are governed by enzyme adsorption to mineral surface. Oxigenation of mineral-bound Fe(II) generates reactive oxygen species (ROS), yet it is unknown whether and how this process alters the activity and functional lifespan of extracellular enzymes. Here, the effect of mineral-bound Fe(II) oxidation on the hydrolytic activity of a cellulose-degrading enzyme ß-glucosidase (BG) was studied using two pre-reduced Fe-bearing clay minerals (nontronite and montmorillonite) and one pre-reduced iron oxide (magnetite) at pH 5 and 7. Under anoxic condition, BG adsorption to mineral surface decreased its activity but prolonged its lifespan. Under oxic condition, ROS was produced, with the amount of •OH, the most abundant ROS, being positively correlated with the extent of structural Fe(II) oxidation in reduced minerals. •OH decreased BG activity and shortened its lifespan via conformational change and structural decomposition of BG. These results suggest that under oxic condition the ROS-induced inhibitory role of Fe(II)-bearing minerals outweighed their adsorption-induced protective role in controlling enzyme activity. These results disclose a previously unknown mechanism of extracellular enzyme inactivation, which have pivotal implications for predicting the active enzyme pool in redox-oscillating environments.

58 GEOSCIENCES↗

NASA NTRS2005

Removing Pathogens Using Nano-Ceramic-Fiber Filters

A nano-aluminum-oxide fiber of only 2 nanometers in diameter was used to develop a ceramic-fiber filter. The fibers are electropositive and, when formulated into a filter material (NanoCeram(TradeMark)), would attract electro-negative particles such as bacteria and viruses. The ability to detect and then remove viruses as well as bacteria is of concern in space cabins since they may be carried onboard by space crews. Moreover, an improved filter was desired that would polish the effluent from condensed moisture and wastewater, producing potable drinking water. A laboratory- size filter was developed that was capable of removing greater than 99.9999 percent of bacteria and virus. Such a removal was achieved at flow rates hundreds of times greater than those through ultraporous membranes that remove particles by sieving. Because the pore size of the new filter was rather large as compared to ultraporous membranes, it was found to be more resistant to clogging. Additionally, a full-size cartridge is being developed that is capable of serving a full space crew. During this ongoing effort, research demonstrated that the filter media was a very efficient adsorbent for DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and endotoxins. Since the adsorption is based on the charge of the macromolecules, there is also a potential for separating proteins and other particulates on the basis of their charge differences. The separation of specific proteins is a major new thrust of biotechnology. The principal application of NanoCeram filters is based on their ability to remove viruses from water. The removal of more than 99.9999 percent of viruses was achieved by a NanoCeram polishing filter added to the effluent of an existing filtration device. NanoCeram is commercially available in laboratory-size filter discs and in the form of a syringe filter. The unique characteristic of the filter can be demonstrated by its ability to remove particulate dyes such as Metanyl yellow. Its particle size is only 2 nanometers, about the size of a DNA molecule, yet the NanoCeram syringe filter is capable of retaining the dyes as the fluid is passed through the syringe, without much back-pressure. Endotoxins, which are contaminants that are part of the residue of destroyed bacteria, can cause toxic shock and are therefore of major concern in pharmaceutical products. The NanoCeram syringe filter is capable of removing greater than 99.96 percent of the endotoxins.

Tepper, Frederick↗

NASA NTRS2000

Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient

Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

Thomas, B. R.↗

DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

NASA NTRS2002

Study of Fluid Flow Control In Protein Crystallization Using Strong Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in 'microgravity', researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined.

Ramachandran, N.↗

NASA NTRS2002

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Ramachandran, Narayanan↗

NASA NTRS2002

Countering Solutal Buoyant Convection with High Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemist, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitant, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity, we have been able to dramatically effect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the current status of the investigation and discuss results from the experimental and modeling efforts.

Ramachandran, N.↗

NASA NTRS2003

Using Strong Magnetic Fields to Control Solutal Convection

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity , we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagnetic salts and solutions in magnetic fields and compare them to analytical predictions.

Ramachandran, N.↗

DOE OSTI2020

Water-Soluble Flexible Organic Frameworks That Include and Deliver Proteins

Four water-soluble hydrazone-based three-dimensional (3D) flexible organic frameworks FOF-1-4 have been synthesized from a semirigid tetracationic tetraaldehyde and four flexible dihydrazides. 1 H NMR spectroscopy indicated the quantitative formation of FOF-1-4 in D 2 O, while dynamic light scattering experiments revealed that, depending on the concentration, these porous frameworks display hydrodynamic diameters ranging from 50 to 120 nm. The porosity of the frameworks is confirmed by ethanol vapor adsorption experiments of the solid samples as well as the high loading capacity for a 2.3 nm porphyrin guest in water. The new water-soluble frameworks exhibit low cytotoxicity and form inherent pores with diameters of 5.3 or 6.7 nm, allowing rapid inclusion of proteins such as bovine serum albumin and green and orange fluorescent proteins, and efficient delivery of the proteins into normal and cancer cells. Flow cytometric analysis reveals percentages of the delivered cells up to 99.8%.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2004

Magnetic Control of Convection during Protein Crystallization

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteract terrestrial gravity. The genera1 objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagentic salts and solutions in magnetic fields and compare them to analyticalpr~ctions.

Ramachandran, N.↗

DOE OSTI2020

Enzyme interactions on lignocellulosic biomass structure

This chapter shows that, although well studied over the years, the cellulose enzymatic hydrolysis process has still many obstacles to be overcome or minimized. High enzyme loading and relatively long incubation periods are generally required for effective cellulose saccharification to be achieved. These limitations make the process unattractive from an economic perspective. Most studies show that the conversion rate of cellulose hydrolysis decreases over reaction time due to several factors that may inhibit, deactivate, or slow the work of enzymes. Some of these factors are inhibition by the product generated; inhibition/deactivation of enzymes by by-products generated during pretreatment (PT); limitation of mass transfer; shear stress by high stirring; air–liquid interfacial area; and unproductive adsorption on lignin or other compounds present in the biomass. The interaction between cellulases and the lignin present in these biomasses has a major negative impact on the process, with the source of the raw material and the type of PT used having a strong influence on it. Finally, the use of nonenzymatic proteins seems to be the best way to mitigate this problem.

20 FOSSIL-FUELED POWER PLANTS↗

DOE OSTI2021

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

36 MATERIALS SCIENCE↗

NASA NTRS2001

Dipole potentials indicate restructuring of the membrane interface induced by gadolinium and beryllium ions

The dipole component of the membrane boundary potential, phi(d), is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Deltaphi(d), and apply it in studies of Be(2+) and Gd(3+) interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Deltaphi(d) is determined as the difference between the changes of the total boundary potential, phi(b), measured by the IFC method in planar lipid membranes and the surface potential, phi(s), determined from the electrophoretic mobility of liposomes. The Gouy-Chapman-Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be(2+) and Gd(3+) to PC membranes, and of Mg(2+) to PS membranes, the values of Deltaphi(b) and Deltaphi(s) are the same, indicative of no change of phi(d). Binding of Gd(3+) to PS-containing membranes induces changes of phi(d) of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Deltaphi(d) is negative (-30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be(2+) or Gd(3+) induces an increase of phi(d) of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd(3+) is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.

Non-NASA Center↗

DOE OSTI2022

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Phage K gp102 Drives Temperature-Sensitive Antibacterial Activity on USA300 MRSA

There is widespread interest in using obligately lytic bacteriophages (“phages”) to treat human bacterial infections. Among Staphylococcus aureus infections, the USA300 lineage is a frequent cause of invasive disease. We observed that phage K, a model S. aureus myophage, exhibits temperature-sensitive growth on USA300 strains, with the wild-type phage providing poorer growth suppression in broth and forming smaller and fainter plaques at 37 °C vs. 30 °C. We isolated 65 mutants of phage K that had improved plaquing characteristics at 37 °C when compared to the parental phage. In all 65 mutants, this phenotype was attributable to loss-of-function (LoF) mutations in gp102, which encodes a protein of unknown function that has homologs only among the Herelleviridae (SPO1-like myophages infecting gram-positive bacteria). Additional experiments with representative mutants consistently showed that the temperature-sensitive plaque phenotype was specific to USA300 MRSA strains and that Gp102 disruption was correlated with improved suppression of bacterial growth in broth and improved antibacterial activity in a mouse model of upper respiratory tract infection. The same genotype and in vitro phenotypes could be replicated in close relatives of phage K. Gp102 disruption did not have a detectable effect on adsorption but did delay cell culture lysis relative to wild-type under permissive infection conditions, suggesting that gp102 conservation might be maintained by selective pressure for more rapid replication. Expression of gp102 on a plasmid was toxic to both an MSSA and a USA300 MRSA strain. Molecular modeling predicts a protein with two helix-turn-helix domains that displays some similarity to DNA-binding proteins such as transcription factors. While its function remains unclear, gp102 is a conserved gene that is important to the infection process of Kayvirus phages, and it appears that the manner in which USA300 strains defend against them at 37 °C can be overcome by gp102 LoF mutations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Electropolymerized-molecularly imprinted polymers (E-MIPS) as sensing elements for the detection of dengue infection

Here, a straightforward in situ detection method for dengue infection was demonstrated through the molecular imprinting of a dengue nonstructural protein 1 (NS1) epitope into an electropolymerized molecularly imprinted polyterthiophene (E-MIP) film sensor. The key enabling step in the sensor fabrication is based on an epitope imprinting strategy, in which short peptide sequences derived from the original target molecules were employed as the main template for detection and analysis. The formation of the E-MIP sensor films was facilitated using cyclic voltammetry (CV) and monitored in situ by electrochemical quartz crystal microbalance (EC-QCM). Surface properties were analyzed using different techniques including atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and polarization modulation-infrared reflection-adsorption (PM-IRRAS). The standard calibration curve (R = 0.9830) was generated for the detection of the epitope, Ac-VHTWTEQYKFQ-NH 2 , with a linear range of 0.2 to 30 μg/mL and detection limit of 0.073 μg/mL. A separate calibration curve (R = 0.9786) was obtained using spiked buffered solutions of dengue NS1 protein, which resulted in a linear range of 0.2 to 10 μg/mL and a detection limit of 0.056 μg/mL. The fabricated E-MIP sensor exhibited long-term stability, high sensitivity, and good selectivity towards the targeted molecules. These results indicated that the formation of the exact and stable cavity imprints in terms of size, shape, and functionalities was successful. In our future work, we aim to use our E-MIP sensors for NS1 detection in real-life samples such as serum and blood.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1998

The Effects of Impurities on Protein Crystal Growth and Nucleation: A Preliminary Study

Kubota and Mullin (1995) devised a simple model to account for the effects of impurities on crystal growth of small inorganic and organic molecules in aqueous solutions. Experimentally, the relative step velocity and crystal growth of these molecules asymptotically approach zero or non-zero values with increasing concentrations of impurities. Alternatively, the step velocity and crystal growth can linearly approach zero as the impurity concentration increases. The Kubota-Mullin model assumes that the impurity exhibits Langmuirian adsorption onto the crystal surface. Decreases in step velocities and subsequent growth rates are related to the fractional coverage (theta) of the crystal surface by adsorbed impurities; theta = Kx / (I +Kx), x = mole fraction of impurity in solution. In the presence of impurities, the relative step velocity, V/Vo, and the relative growth rate of a crystal face, G/Go, are proposed to conform to the following equations: V/Vo approx. = G/Go = 1 - (alpha)(theta). The adsorption of impurity is assumed to be rapid and in quasi-equilibrium with the crystal surface sites available. When the value of alpha, an effectiveness factor, is one the growth will asymptotically approach zero with increasing concentrations of impurity. At values less than one, growth approaches a non-zero value asymptotically. When alpha is much greater than one, there will be a linear relationship between impurity concentration and growth rates. Kubota and Mullin expect alpha to decrease with increasing supersaturation and shrinking size of a two dimensional nucleus. It is expected that impurity effects on protein crystal growth will exhibit behavior similar to that of impurities in small molecule growth. A number of proteins were added to purified chicken egg white lysozyme, the effect on crystal nucleation and growth assessed.

Schall, Constance A.↗

DOE OSTI2021

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

Background: Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass. Results: In this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation. Conclusions: Enzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

09 BIOMASS FUELS↗