Journal: PLoS ONE

Article Title: A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

doi: 10.1371/journal.pone.0043817

Figure Lengend Snippet: (A) Circular dichroism spectra averaged from four scans and corrected for the 10 mM sodium phosphate buffer are shown. Both triadin peptides (triadin 200–231 (black trace) and triadin 200–232 (grey trace); 0.03 mg/ml) show a negative peak at ∼197 nm, consistent with an intrinsically disordered structure. A positive peak at ∼190 nm and negative peaks at ∼208 and ∼223 nm (indicative of α-helical secondary structure) or a positive peak at ∼195 nm and a negative peak at ∼217 nm (indicative of β-sheet secondary structure), are absent. (B) Western blot, following streptavidin-agarose affinity chromatography, showing the association of RyR1 with biotin tagged triadin peptide. The upper half of the membrane was probed with anti-RyR1 antibody and the lower half was probed with Streptactin-HRP conjugate to identify the biotin tagged peptides. Lane 1 protein sample eluted from streptavidin-agarose incubated with RyR1 alone; Lane 2 purified RyR1 alone (control); Lane 3 biotin tagged triadin 200–231 peptide alone (control); Lanes 4 and 5 protein sample eluted from streptavidin-agarose affinity chromatography, where streptavidin-agarose was incubated with biotin tagged triadin 200–232 or triadin 200–231 peptide respectively, prior to incubation with RyR1.

Article Snippet: The RyR1 4871–4910 peptide, containing the putative Trisk 95 binding domain on the RyR1 , was produced and HPLC purified by GL Biochem (Shanghai) Ltd (Shanghai, China).

Techniques: Circular Dichroism, Western Blot, Affinity Chromatography, Membrane, Incubation, Purification, Control

Journal: PLoS ONE

Article Title: A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

doi: 10.1371/journal.pone.0043817

Figure Lengend Snippet: (A) Running histogram of a typical bilayer experiment. Open probability ( P o ) was measured every 30 s throughout the lifetime of the experiment before and after the addition of 63 nM and then 252 nM triadin 200–231 peptide (arrows) at +40 mV (light grey bins) and −40 mV (dark grey bins). Data averages for each condition are shown as horizontal broken lines for +40 mV and −40 mV, and median is presented as a horizontal solid line. (B, C) 3 s traces of purified RyR1 channel activity. Activity was recorded at +40 mV (left) where channels are opening upward from zero current (c, continuous line) to maximum open conductance (o, broken line) and at −40 mV (right), where channel openings are downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). Top panel – control recording of purified RyR1 prior to the addition of triadin peptide; middle and bottom panel – after the addition of 63 nM and 252 nM triadin peptide to the trans chamber. (B) Shows addition of triadin 200–232 peptide, and (C) shows addition of triadin 200–231 . Control P o in the absence of peptide was 0.46±0.12 at +40 mV and 0.38±0.17 at −40 mV (for triadin 200–232 ), and 0.14±0.07 at +40 mV and 0.29±0.17 at −40 mV (for triadin 200–231 ).

Article Snippet: The RyR1 4871–4910 peptide, containing the putative Trisk 95 binding domain on the RyR1 , was produced and HPLC purified by GL Biochem (Shanghai) Ltd (Shanghai, China).

Techniques: Purification, Activity Assay, Control

Journal: PLoS ONE

Article Title: A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

doi: 10.1371/journal.pone.0043817

Figure Lengend Snippet: (A) Average data for open probability ( P o ) in presence of triadin 200–232 and triadin 200–231 (n = 10), data from both peptides combined (see results text, collectively termed triadin peptide, n = 11–20) for each of the following parameters; open time ( T o ), close time ( T c ) and open frequency ( F o ), collected at −40 mV and +40 mV. All data is expressed as relative mean data (Log rel (parameter)). Relative mean P o (log rel P o ) is the average of differences between the log 10 of the P o in the presence of either 63 nM or 252 nM triadin peptide (log 10 P o Pep ) and log 10 of the control P o (log 10 P o Con ) for each channel. Log rel T o is log 10 T o Pep -log 10 T o Con , log rel T c is log 10 T c Pep -log 10 T c Con and log rel F o is log 10 F o Pep -log 10 F o Con . (B) [ 3 H]ryanodine binding to purified RyR1 in the absence and presence of 63 nM of triadin peptide. [ 3 H]ryanodine binding is measured as pmol ryanodine/mg RyR1. Data is expressed relative to binding recorded in the absence of peptides (rel ryanodine binding). Significance (p≤0.05) is indicated for each concentration compared to activity recorded prior to addition of peptide (*).

Article Snippet: The RyR1 4871–4910 peptide, containing the putative Trisk 95 binding domain on the RyR1 , was produced and HPLC purified by GL Biochem (Shanghai) Ltd (Shanghai, China).

Techniques: Control, Binding Assay, Purification, Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

doi: 10.1371/journal.pone.0043817

Figure Lengend Snippet: Channel open probability parameter values for native and purified RyR1 measured in the presence and absence of 63 nM Triadin peptide and 63 nM full length Trisk 95.

Article Snippet: The RyR1 4871–4910 peptide, containing the putative Trisk 95 binding domain on the RyR1 , was produced and HPLC purified by GL Biochem (Shanghai) Ltd (Shanghai, China).

Techniques: Purification

Journal: PLoS ONE

Article Title: A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

doi: 10.1371/journal.pone.0043817

Figure Lengend Snippet: (A–B) 3 s traces of RyR1 channel activity at −40 mV. Channels are opening downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). (A) RyR1 ΔM 1,2,3 mutants (with Trisk 95 binding residues mutated) in absence of triadin peptide and in the presence of trans 63 nM and 252 nM peptide. (B) Native RyR1 in the absence and presence of trans 63 nM triadin peptide. Average open probability ( P o ) of data, collected at −40 mV and +40 mV (n = 6–10) is displayed below each trace. (C) [ 3 H]ryanodine binding to purified RyR with triadin binding blocking peptide (RyR1 4871–4910 ) in the absence and presence of 63 nM triadin peptide. [ 3 H]ryanodine binding is measured as pmol ryanodine/mg RyR1 and data is expressed relative to binding recorded in the absence of either peptide (rel ryanodine binding). All values are presented as relative to control. Asterisks (*) indicate a significant difference (p≤0.05) in binding from RyR1 control (no peptide); crosshatch (#) indicates a significant difference (p≤0.05) in binding from RyR1 in the presence of RyR1 4871–4910 .

Article Snippet: The RyR1 4871–4910 peptide, containing the putative Trisk 95 binding domain on the RyR1 , was produced and HPLC purified by GL Biochem (Shanghai) Ltd (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Purification, Blocking Assay, Control