Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Magnesium-induced folding of SL ribozyme constructs. Peak E shifts from the Gaussian fits of the FRET histograms at Mg2+ concentrations from 0.01 mM to 1 M for the SL (▪) and WT (⋄) 2WJ, 3WJ, and 4WJ ribozymes (parts A, B, and C, respectively). Peak E values for a control donor-acceptor labeled DNA molecule with dyes separated by 14 base pairs are shown for comparison as solid triangles in the 3WJ graph in part B.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Control, Labeling, Comparison

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Ratiometric spFRET data for the WT 4WJ ribozyme. (A) Example of donor (black) and acceptor (gray) emission bursts observed in spFRET experiments. A time trace is presented for the WT 4WJ ribozyme at a concentration of 100 pM in 50 mM Tris/HCl buffer with 0.1 mM Mg2+ at room temperature, and the data integration time per point was 0.5 ms. Fluorescent donor and acceptor bursts are observed above background. A buffer (background) time trace is also shown for comparison. Only bursts above a threshold of 40 counts (sum of donor and acceptor signals) were used to construct FRET efficiency histograms. (B) FRET efficiency histogram derived from calculation of FRET efficiencies from each accepted donor/acceptor event above a threshold of 40 counts, showing the docked and undocked subpopulations (see text), as well as the zero-E peak. The solid lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Concentration Assay, Comparison, Construct, Derivative Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: FRET efficiency histogram for a G11I mutant of the 4WJ ribozyme at 0.1 mM Mg2+, using the same conditions as for data presented in Fig. 3. Solid lines show fits using Gaussian functions. The relative area of the high efficiency (docked) peak is markedly reduced relative to the wild-type 4WJ ribozyme (Fig. 3), reflecting a destabilized tertiary structure for the mutant.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Mutagenesis

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Schematic representation of WT ribozyme constructs used for spFRET studies (upper panel). FRET efficiency histograms (bars) for wild-type 2WJ, 3WJ, and 4WJ hairpin ribozymes, showing undocked and docked ribozyme subpopulations at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Single-loop 2WJ, 3WJ, and 4WJ ribozyme constructs (upper panel) and corresponding FRET efficiency histograms at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Kinetic information from peak shape analysis of spFRET histograms. (A) Overlay of FRET efficiency histograms for SL (bold solid line) and WT 4WJ (data as bar graph, Gaussian fit as solid line) ribozymes at 10 mM Mg2+, emphasizing the asymmetric peak shape for the SL 4WJ. The error bars represent single standard deviations. (B) Overlay of FRET histograms from two-state simulations (lines) and experimental data (bars; errors represent single standard deviations) for the SL 4WJ at 10 mM Mg2+. The extended-undocked (EU)–quasi-docked (QD) equilibrium scheme is shown in the inset. Simulated histograms are shown for forward/reverse (k1/k−1) rate constants of 20 × 103/6.8 × 103 s−1 (bold solid line, 1), 41 × 103/14 × 103 s−1 (bold dashed line, 2), and 10 × 103/3.4 × 103 s−1 (bold dash-dot-dashed line, 3) with a corresponding equilibrium constant of 3 in favor of the quasi-docked state in all cases. See text for details.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques:

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: PAS-mutant hERGs are sorted for lysosomal delivery from the cell surface. ( a ) hERG is targeted to LAMP1-positive endo-lysosomal compartments. Endocytic WT, M124R and C64Y hERG pool labelled by Ab capture (15 min at 37 °C) and remaining cell-surface hERG blocked with unconjugated secondary F(ab′) 2 (1 h on ice). Cells then chased at 37 °C for 3 h prior to fixation. Lysosomal compartments labelled with LAMP1 pAb. hERG (green) and LAMP1 (magenta) staining visualized by LCFM. Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 5 µm, right) images shown. Magnified area indicated by white box. Analysis of additional mutants (F29L, R56Q, T65P) in Supplementary Fig. . ( b ) Representative distribution of vesicular pH for WT and T65P hERG containing endocytic vesicles following 3 h chase. Overlay of multi-Gaussian peak-fits shown and mean pH ± SD indicated. N indicates total number of vesicles analyzed in a representative experiment. ( c ) PAS-mutations accelerate hERG endo-lysosomal delivery kinetics. Mean luminal pH of vesicles containing WT or T65P hERG measured by FRIA. Anti-HA Ab and FITC-Fab were bound on ice and FRIA was performed after 1- to 6-h chase. ( d ) Mean luminal pH of vesicles containing WT and PAS-mutant hERG following 3 h chase. ( e , f ) Lysosomal activity contributes to degradation of mature hERG proteins. Metabolic stability of WT and PAS-mutants hERG evaluated by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). V-ATPase inhibition with Bafilomycin A1 (BafA1, 200 nM), or proteasome inhibition with Bortezomib (Bort, 3 µM) or Ixazomib (Ixa, 3 µM) attenuated the rapid degradation of PAS-mutants. Mature complex-glycosylated (~155 kDa) and ER-resident core-glycosylated (~135 kDa) hERG indicated by solid and empty arrows, respectively. Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See methods and materials for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Mutagenesis, Staining, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: Peripheral quality control engagement is dependent on conformational destabilization. ( a ) Mature hERG is destabilized at elevated temperature. Metabolic stability of mature WT, PAS-mutant (F29L and T65P) or temperature-rescued G601S (48 h at 26 °C, rG601S) hERG evaluated at 37 °C or 41 °C by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. ( b ) Turnover kinetics of mature WT and F29L hERG fit using single-exponential decay functions. Similar results obtained for T65P and rG601S hERG (Supplementary Fig. ). ( c ) Turnover rate-constants determined by curve fitting as in ( b ) and expressed as fold increase relative to 37 °C. ( d ) Pharmacological correction of hERG folding restores cell-surface stability. PM-turnover of WT and select PAS-mutants hERG measured by cell-surface ELISA following overnight (16 h) E4031 treatment (10 µM). ( e ) Pharmacochaperone treatment improves folding of nascent hERG at the ER but does not promote refolding of mature channels at the PM. Internalization of WT and select PAS-mutant hERG measured by PM-ELISA following acute (1 h) or overnight (16 h) E4031 pre-treatment (10 µM). ( f ) Delivery of PM-labelled T65P hERG to LAMP1-positive compartments evaluated by LCFM following 3 h chase. Lysosomal delivery is prevented by overnight pre-treatment with E4031 (10 µM). Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 2 µm, right) images shown. Magnified area indicated by white box. Analysis of WT and additional PAS-mutants in Supplementary Fig. . ( g ) Pharmacochaperone pre-treatment prevents endo-lysosomal trafficking of T65P hERG. Representative histogram of T65P hERG vesicular pH following 3 h chase. Overlay of multi-Gaussian peak-fits (mean ± SD) shown. N indicates total number of vesicles evaluated. ( h ) Mean luminal pH of hERG-containing endocytic vesicles measured by FRIA following overnight treatment with E4031 (10 µM) and 3 h chase at 37 °C. ( i ) Subset of temperature-rescued PAS-mutants are resistant to unfolding at physiological temperature. Internalization of WT and PAS-mutant hERG measured by PM-ELISA following low-temperature rescue (30 °C for 24 h) and unfolding (37 °C for 2 h). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Control, Mutagenesis, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Graphics & Modelling

Article Title: Evaluation of anti-cancer and anti -covid-19 properties of cationic pentapeptide Glu-Gln-Arg-Pro-Arg, from rice bran protein and its d -isomer analogs through molecular docking simulations

doi: 10.1016/j.jmgm.2021.107999

Figure Lengend Snippet: A) Normalized amide I and amide II bands of FTIR spectra of the peptide EQRPR and its d -isomer analogs. Thick black and thin white lines represent experimental and best fitting spectra, respectively. The best fitting spectra are results of eight Gaussian component fitting (See Methods for details). Two Gaussian components around 1730 cm −1 and 1545 cm −1 that represent the contribution from side-chains and amid II, respectively, were not considered in the calculation of the secondary structures of the peptides. B) and C) Secondary structure content of the peptide EQRPR and its d -isomer analogs. A and B are data derived from FTIR and CD spectroscopies, respectively.

Article Snippet: Fitting the spectra to multiple Gaussian components was performed using OriginLab software (OriginLab Corp., Northampton, MA).

Techniques: Derivative Assay