multiple gaussian fit  (GraphPad Software Inc)

Journal: Biophysical Journal

Article Title: Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B

doi: 10.1016/j.bpj.2019.10.006

Figure Lengend Snippet: Amplitude characteristics of spontaneous IA(glu) events are consistent with multiquantal release. (A) Spontaneous multiquantal events were observed among many uniquantal events in rods voltage clamped at −70 mV. (B) Shown is a representative amplitude histogram of spontaneous rod IA(glu) events (n = 325) fit with a multiple Gaussian function. The inset shows a representative segment of the recording from which the histogram was derived. By assuming that the mean ± SD of the initial peak represents a single quantum, quantal content for this cell was calculated as a weighted average of the areas under the curve and found to be 1.69. (C) Amplitude of IA(glu) events (n = 339) was strongly correlated with event charge transfer (R = 0.99) with nonzero slope (F-test, p < 0.0001).

Article Snippet: Events within one SD of the mean of the first peak were considered “uniquantal,” and remaining larger events were considered “multiquantal.” Quantal content was calculated from weighted averages of the area under the curves from a multiple Gaussian fit (GraphPad Prism 4).

Techniques: Derivative Assay

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Using AFM to detect the presence, absence, or the blocking of isopeptide bonds in the Spy0128 construct. (A) The native intramolecular isopeptide bond in Spy0128 prevents the protein from unfolding and extending under force. Unfolding sawtooth pattern obtained by stretching the unmodified Spy0128 construct. These traces only show the three I91 fingerprints with short initial extensions (Li, defined as the extension to the first I91 unfolding) and contour length increments of 29 nm, measured using fits of the worm-like chain model of polymer elasticity (solid lines). Data are from ref. 24. (B) Mutant Spy0128 (E258A) where the isopeptide bond is not formed. In this case, the sawtooth patterns show two additional high-force unfolding events that always follow the I91 fingerprints and have large contour length increments of ∼50 nm. Data from ref. 24. (C) Spy0128 constructs modified by the isopeptide blocker show a very different type of sawtooth pattern, with long initial extensions marked by a series of random low-force peaks observed always before the I91 fingerprint. (D) Histogram of initial extensions (Li) measured from sawtooth patterns obtained from Spy0128 constructs that were coexpressed with the isopeptide blocker (n = 698). A Gaussian fit (solid line) marks three distinct peaks at 41, 80, and 130 nm. The peak at 41 nm corresponds to unmodified Spy0128 constructs. The peaks at 80 and 130 nm closely correspond to the extensions predicted if one or both isopeptide bonds were blocked and the modified protein was unable to fold.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Blocking Assay, Construct, Polymer, Mutagenesis, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Mechanical properties of HaloTag-purified Spy0128 constructs. Using Promega paramagnetic beads, we enrich the fraction of proteins that have been modified by the blocking peptide. The proteins are subsequently detached from the beads using the TEV protease. From these purified proteins we obtained three types of sawtooth patterns: (A) Unmodified Spy0128 constructs with short initial extensions (Li < 50 nm) and (B) showing a single modification (Li ∼ 50–100 nm) or (C) two modifications (Li > 100 nm). (D) Histogram of initial extensions (n = 453). A Gaussian fit identifies three peaks at 34, 86, and 135 nm. (E) Two-dimensional histogram showing the density of unfolding forces (FU) versus the contour length increments (∆Lc) for the isopeptide-blocked Spy0128. Modified pilin proteins unfold at forces below 100 pN and do not show any well-defined unfolding pathway. Remarkably, 14% of the traces (n = 40) show long initial extensions without any unfolding event, suggesting that the modified Spy0128 extends as an unstructured polymer. (F) Mutant Spy0128 E258A unfolds through well-defined unfolding pathways with high mechanical stability, either through a single event with FU ∼ 300 pN or through an intermediate with FU1 ∼ 180 pN and FU2 ∼ 110 pN. Data from ref. 24.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Purification, Construct, Modification, Blocking Assay, Polymer, Mutagenesis