Nanopore Sensing of Protein Folding

Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding statesattractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein’s folding state. Our all-atom molecular dynamics (MD) simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proximity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding–unfolding transition is detectable despite the orientational and conformational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding–unfolding transitions in real time and report on the structure of folding intermediates

Optimal temperature, thermal tolerance, optimal pH and pH stability of Atm.

<p>A. The optimal temperature was determined at various temperatures (30°C–70°C) and at pH 4.5. B. The thermos-stability of Atm was investigated by pre-incubation of the enzyme solutions for 10 min to 12 hours in the absence of substrate at pH4.5, at different temperatures (30°C–70°C) and residual laccase activities were determined. C. The optimal pH was evaluated at 30°C over a pH range of 2.0–9.0. D. pH stability of Atm was evaluated at 30°C over a pH range of 2.0–9.0. The activity measured at pH 4.5 and at 30°C was considered as 100%. Error bars represent the standard errors of the means.</p

Alignment of the four copper-binding sites in laccases-related proteins.

<p>Conserved amino acids are highlight in black. T₁, T₂, T₃A, and T₃B indicate the putative corresponding type 1, 2, and 3 copper centers. For each copper center, arrows point to the copper-binding amino acid residues. The horizontal line separates bacterial proteins (above the line) from fungal proteins (below the line). <i>S</i>. <i>griseus</i>: <i>Streptomyces griseus</i>; <i>E</i>. <i>coli</i>: <i>Escherichia coli</i> K-12; <i>T</i>. <i>thermophilus</i>: <i>Thermus thermophilus</i> HB27; <i>B</i>. <i>subtilis</i>: <i>Bacillus subtilis</i>; <i>B</i>. <i>licheniformis</i>: <i>Bacillus licheniformis</i>; <i>B</i>. <i>halodurans</i>: <i>Bacillus halodurans</i> C-125; <i>B</i>. <i>fuckeliana</i>: <i>Botryotinia fuckeliana</i>; <i>T</i>. <i>villosa</i>: <i>Trametes villosa</i>; <i>A</i>. <i>niger</i>: <i>Aspergillus niger</i>.</p

Effect of heavy metals, organic solvents and enzyme inhibitors on enzymatic stability of Atm.

<p>DMSO: dimethyl sulfoxide; MeOH: methanol; EtOH: ethanol; SDS: sodium dodecyl sulfate; DTT: dithiothreitol; EDTA: ethylene diamine tetraacetie acid; NaN₃: sodium azide. Error bars represent the standard errors of the means.</p

SDS-PAGE and Western blot analysis of Atm.

<p>A. SDS-PAGE analysis. M: Protein markers; Lane 1: cell extract; Lane 2: unbound proteins in the flow through from the column; Lane 3: the fraction from the wash buffer containing 40 mM imidazole; Lane 4: the fraction from the wash buffer containing 100 mM imidazole; Lane 5: the fraction from the elution buffer containing 500 mM imidazole. B. Western blot analysis. M: Protein markers; lane 1: protein extracts from pG-KJE8/BL21 with pCold I—atm; lane 2: protein extracts from pG-KJE8/BL21 with pCold I, as a negative control; lane 3, protein extracts from pG-KJE8/BL21, as an additional negative control.</p

Enzymological Characterization of Atm, the First Laccase from <i>Agrobacterium sp</i>. S5-1, with the Ability to Enhance <i>In Vitro</i> digestibility of Maize Straw

<div><p>Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (<i>atm</i>) in a ligninolyitic isolate <i>Agrobacterium sp</i>. S5-1 from soil humus was identified and heterologously expressed in <i>Escherichia coli</i>. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co³⁺, Mn²⁺, Cu²⁺ and Ni²⁺, 20 mM) and all tested organic solvents. Furthermore, Atm significantly (<i>p</i><0.05) increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (<i>p</i><0.05) enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved <i>in vitro</i> digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol.</p></div

Analysis of factors impacting the infection efficiency of NDV-pseudotyped virus.

<p>A panel of mutant and wild-type HN and F combinations were co-transfected into 293 T cells with the HIV-Luc vector. Pseudoviruses in the culture medium were harvested 48 h later and used to infect 293 T cells in 96-well plates. The luciferase activity of the infected cells was detected 48 h post infection and normalized with the titer of p24 in the pseudoviruses. All results are shown as means ± SD from four independent experiments. “**” indicated <i>P</i><0.01.</p

Analysis of NDV-pseudotyped HIV-Luc viruses.

<p>HIV-Luc vector was co-transfected into 293 T cells with HN and F or VSV-G. Pseudoviruses were harvested 48 h post transfection and used for the analysis of their incorporation and infection. (A) and (B) The pseudotyped HIV-Luc viruses by HN and F or VSV-G envelope proteins were purified with ultracentrifugation and coated to the ELISA plate. F and HN proteins incorporated into pseudotyped HIV-Luc virus were probed by anti-HN mAb (A) or anti-F mAb (B); (C) The pseudotyped HIV-Luc viruses by HN and F or VSV-G envelope proteins were used to infect 293 T cells in 96-well plates. 48 h later, luciferase activity of infected cells was detected and normalized with titer of p24 protein in pseudoviruses. HN + F: NDV-pseudotyped HIV-Luc virus; VSV-G: VSV-G-pseudotyped HIV-Luc virus; control: uninfected cells. All results are shown as means ± SD from three independent experiments. “**” indicated <i>P</i><0.01.</p

Neutralization assay of NDV-pseudotyped HIV-Luc virus by the anti-NDV sera.

<p>Sera against NDV were two-fold serially diluted with DMEM. 100 µl titrated NDV-pseudotyped HIV-Luc viruses were mixed with an equal volume of diluted anti-NDV sera. The mixtures were added to 293 T cells in 96-well plates after incubating at room temperature for 1 h. (A) Neutralization of NDV-pseudotyped virus by the immune sera against NDV. Negative serum and cell control refer to the cells infected with a mixture of negative serum from chickens with NDV-pseudotyped virus and cells without pseudovirus infection, respectively; (B) Neutralization efficiency of NDV-pseudotyped HIV-Luc virus by the three anti-NDV sera at an indicated dilution. Results are shown as means ± SD from four independent infections.</p

Comparison of NT titers determined by NDV-pseudotyped HIV-Luc virus with those by VN test.

<p>(A) NT titers of sixteen immune sera against NDV were determined by NDV-pseudotyped HIV-Luc viruses and VN test, respectively. The obtained data were stacked on the column. White and black column were NT titers determined by VN test and NDV-pseudotyped HIV-Luc viruses, respectively. (B) Correlation between NT titers determined by NDV-pseudotyped HIV-Luc virus and those obtained with VN test. The correlation coefficient was 0.92. NT titer (log₂) values determined by NDV-pseudotyped HIV-Luc virus plotted between 6.5 and 9 on the x-axis scale, whereas, those by VN test plotted between 5 and 8 on the y-axis scale.</p